Angiogenic agent and method of manufacturing the same

ABSTRACT

An object of the present invention is to provide an angiogenic agent that can sufficiently exhibit an angiogenic effect due to mesenchymal stem cells in a state where the angiogenic agent does not allow permeation of host cells while being protected from immune rejection, and a method for method for manufacturing the same. According to the present invention, an angiogenic agent including a mesenchymal stem cell (A); and an immunoisolation membrane (B) that encloses the mesenchymal stem cell is provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of PCT International Application No.PCT/JP2018/032162 filed on Aug. 30, 2018, which claims priority under 35U.S.C § 119(a) to Japanese Patent Application No. 2017-165416 filed onAug. 30, 2017 and Japanese Patent Application No. 2018-032351 filed onFeb. 26, 2018. Each of the above application(s) is hereby expresslyincorporated by reference, in its entirety, into the presentapplication.

REFERENCE TO SEQUENCE LISTING SUBMITTED VIA EFS-WEB

This application includes an electronically submitted sequence listingin .txt format. The .txt file contains a sequence listing entitled“2870-0748PUS 1_ST25.txt” created on Apr. 15, 2020 and is 12,275 bytesin size. The sequence listing contained in this .txt file is part of thespecification and is hereby incorporated by reference herein in itsentirety.

BACKGROUND OF THE INVENTION 1. Field of the Invention

The present invention relates to an angiogenic agent including amesenchymal stem cell and an immunoisolation membrane enclosing themesenchymal stem cell. The present invention further relates to a methodfor manufacturing an angiogenic agent.

2. Description of the Related Art

Recently, research on regenerative medicine and cell treatment usingmesenchymal stem cells (MSC) has been actively conducted. For example,in JP2009-007321A, a method for preventing/treating inflammatory boweldisease by intravenously administering MSC is disclosed. Severalclinical studies and clinical trials also have been conducted. However,it has been found that a practically sufficient therapeutic effectcannot be exhibited (Aliment Pharmacol Ther 2017; 45: 205-221). Inaddition, JP2016-138092A discloses a method for treating a liver diseaseby administering MSCs cultured in low oxygen. Furthermore, JP5606008Bdiscloses that MSCs cultured in a simulated microgravity environment aresuitable for treating central nervous system diseases. Furthermore,angiogenesis use applications for ischemic diseases have beenextensively studied, and for example, JP5290281B discloses a therapeuticagent for ischemic limb diseases which includes adipose-derivedmesenchymal stem cells.

In consideration of industrial use, there is a problem of acquiring MSCsindustrially. As a solution to this problem, using cells of otherpersons (allogeneic cells) instead of cells of a patient himself orherself is said to be realistic, and many studies using allogeneic MSCshave been carried out in actual clinical studies and clinical trials.However, similar to the problem in organ transplantation from the past,in treatment using allogeneic cells, there is a major problem oftransplanted allogeneic MSCs being rejected and eliminated due to immunereaction of a patient himself or herself. MSCs themselves are said tohave a property by which the MSCs are relatively unlikely to be rejectedby the immune system, but in reality, it is obvious that the MSCs arerejected, and they are eliminated from a body over time. Using animmunosuppressant agent in combination is one solution as in the case oforgan transplantation. However, side effects due to theimmunosuppressant agent and permanent use thereof are a heavy burden,and therefore it is not a desirable solution.

Accordingly, there is a demand for a method for evading an immunereaction without using an immunosuppressant agent. As one method, aselectively permeable membrane called an immunoisolation membrane hasbeen studied as a device for pancreatic islet transplantation which isassumed to be mainly used for treating diabetes. For example,JP1995-508187A (JP-H07-508187A) discloses an immunoisolation membranedevice for transplanting a pancreatic islet that produces insulin, butthe document does not disclose a method for transplanting MSCs.

In addition, JP2008-541953A discloses an implantable device thatprovides a bioactive substance to a subject in need thereof, the deviceincluding a microvascular structure in contact with a biocompatiblesemipermeable pouch that encapsulates one or more kinds of cell capableof producing a bioactive substance. The device of JP2008-541953A is forimproving viability and function of transplanted cells, and the documentdiscloses that the device may be an immunoisolation device.

SUMMARY OF THE INVENTION

An object of the present invention is to provide an angiogenic agentthat can sufficiently exhibit an angiogenic effect due to mesenchymalstem cells in a state where the angiogenic agent does not allowpermeation of host cells while being protected from immune rejection,and a method for method for manufacturing the same.

As a result of intensive studies to achieve the above-described object,the inventors of the present invention have found that, by using animmunoisolation membrane that encloses mesenchymal stem cells, it ispossible to provide an angiogenic agent that can sufficiently exhibit anangiogenic effect due to the mesenchymal stem cells in a state where theangiogenic agent is protected from immune rejection, and therefore havecompleted the present invention.

That is, according to the present invention, the following inventionsare provided.

(1) An angiogenic agent comprising: a mesenchymal stem cell (A); and animmunoisolation membrane (B) that encloses the mesenchymal stem cell.

(2) The angiogenic agent according to (1), in which the mesenchymal stemcell is an adipose-derived mesenchymal stem cell or abone-marrow-derived mesenchymal stem cell.

(3) The angiogenic agent according to (1) or (2), in which theimmunoisolation membrane is a porous membrane including a polymer.

(4) The angiogenic agent according to (3), in which a minimum porediameter of the porous membrane is 0.02 μm to 1.5 μm.

(5) The angiogenic agent according to (3) or (4), in which a thicknessof the porous membrane is 10 μm to 250 μm.

(6) The angiogenic agent according to any one of (3) to (5), in which,within an inner side of the porous membrane, a layered compact portionin which a pore diameter is minimized is present, and a pore diametercontinuously increases in a thickness direction from the compact portiontoward at least one surface of the porous membrane.

(7) The angiogenic agent according to (6), in which a thickness of thecompact portion is 0.5 μm to 30 μm.

(8) The angiogenic agent according to any one of (3) to (7), in which aratio of a maximum pore diameter to a minimum pore diameter of theporous membrane is 3.0 to 20.0.

(9) The angiogenic agent according to any one of (3) to (8), in whichthe porous membrane contains at least one kind of polysulfone andpolyvinylpyrrolidone.

(10) The angiogenic agent according to any one of (1) to (9), in whichthe mesenchymal stem cell is contained as a cell structure whichincludes a plurality of biocompatible polymer blocks and a plurality ofmesenchymal stem cells of at least one type, and in which at least oneof the biocompatible polymer blocks is disposed in gaps between theplurality of mesenchymal stem cells.

(11) The angiogenic agent according to (10), in which a size of one ofthe biocompatible polymer blocks is 20 μm or more and 200 μm or less.

(12) The angiogenic agent according to (10) or (11), in which in thebiocompatible polymer block, a biocompatible polymer is cross-linked byheat, ultraviolet rays, or an enzyme.

(13) The angiogenic agent according to any one of (10) to (12), in whichthe biocompatible polymer block has an amorphous shape.

(14) The angiogenic agent according to any one of (10) to (13), in whichthe cell structure includes 0.0000001 μg to 1 μg of biocompatiblepolymer blocks per cell.

(15) A method for manufacturing the angiogenic agent according to anyone of (1) to (14), the method comprising a step of enclosingmesenchymal stem cells with an immunoisolation membrane.

Furthermore, according to the present invention, the followinginventions are provided.

(16) An angiogenesis method comprising a step of transplanting, to asubject in need of angiogenesis, a cell transplant device including amesenchymal stem cell (A) and an immunoisolation membrane (B) thatencloses the mesenchymal stem cell.

(17) A cell transplant device which is used for angiogenesis procedure,the device comprising a mesenchymal stem cell (A); and animmunoisolation membrane (B) that encloses the mesenchymal stem cell.

(18) Use of a cell transplant device to manufacture an angiogenic agent,in which the cell transplant device includes a mesenchymal stem cell(A); and an immunoisolation membrane (B) that encloses the mesenchymalstem cell.

SUMMARY OF THE INVENTION

The angiogenic agent of the present invention is useful for promotingangiogenesis by transplanting allogeneic or xenogeneic mesenchymal stemcells (MSCs) which cause immune rejection. According to the presentinvention, an angiogenic effect due to MSCs can be sufficientlyexhibited while protecting the MSCs from host cells. Furthermore,according to the present invention, new blood vessels can be locallygenerated around the angiogenic agent at a high efficiency.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a liquid temperature profiling of an experimentdescribed in Condition A.

FIG. 2 illustrates a liquid temperature profiling of an experimentdescribed in Condition B.

FIG. 3 illustrates a liquid temperature profiling of an experimentdescribed in Condition C.

FIG. 4 illustrates a SEM photograph of a cross section of a porousmembrane of Reference Example 8.

FIG. 5 illustrates a pore diameter distribution in a thickness directionof the porous membrane of Reference Example 8.

FIG. 6 illustrates a method for producing a cell transplant device (onlyan immunoisolation membrane).

FIG. 7 illustrates a SEM photograph of a cross section of a porousmembrane of Reference Example 10.

FIG. 8 illustrates a pore diameter distribution in a thickness directionof the porous membrane of Reference Example 10.

FIG. 9 illustrates a SEM photograph of a cross section of a porousmembrane of Reference Example 12.

FIG. 10 illustrates a pore diameter distribution in a thicknessdirection of the porous membrane of Reference Example 12.

FIG. 11 illustrates a tissue specimen into which a cell transplantdevice including a cell structure is transplanted.

FIG. 12 illustrates a tissue specimen into which only a cell structureis transplanted.

FIG. 13 illustrates a tissue specimen into which a cell transplantdevice (only an immunoisolation membrane) is transplanted.

FIG. 14 illustrates measurement results of a total area of blood vesselper field of view.

FIG. 15 illustrates measurement results of the number of blood vesselsper field of view.

FIG. 16 illustrates measurement results of a total area of blood vesselper field of view.

FIG. 17 illustrates measurement results of the number of blood vesselsper field of view.

FIG. 18 illustrates a tissue specimen of a C57BL/6 mouse into which acell transplant device including a cell structure, or a cell transplantdevice (only an immunoisolation membrane) is transplanted.

FIG. 19 illustrates a tissue specimen of a C57BL/6 mouse into which onlya cell transplant device is transplanted.

FIG. 20 illustrates measurement results of a total area of blood vesselper field of view.

FIG. 21 illustrates measurement results of the number of blood vesselsper field of view.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Hereinafter, embodiments for implementing the present invention arespecifically described. The expression “to” in the present specificationrefers to a range including numerical values described before and afterthe expression as a minimum value and a maximum value, respectively.

An angiogenic agent of the embodiment of the present invention includesa mesenchymal stem cell (A), and an immunoisolation membrane (B) thatencloses the mesenchymal stem cell.

The angiogenic agent of the embodiment of the present invention canlocally generate new blood vessels around the angiogenic agent, and canexert a long-term therapeutic effect due to transplanted cells. Theangiogenic agent of the embodiment of the present invention itself doesnot include microvessels, and is different in this respect from thedevice disclosed in JP2008-541953A. In consideration of industrial use,it is realistic to transplant cells or tissues of other persons.However, in the component of JP2008-541953A, a microvessel itself is atarget of rejection due to immune reaction of a host. This isparticularly noticeable in a case where a microvessel includes cells,and in consideration of this phenomenon, JP2008-541953A discloses use ofallogeanic/syngeneic animals. However, allogeanic/syngeneic animals areestablished only between animals genetically controlled as experimentalanimals, and in practice, for example, there is no syngeneic persons ina case of targeting humans, and therefore the component of this documentcannot be used. On the other hand, the angiogenic agent of the presentinvention is greatly different in that it can be used without suchrestrictions because it does not contain externally-derived microvessel,and a new blood vessel is created by host cells.

<Cell>

Cells used in the present invention are mesenchymal stem cells (MSCs).The origin of the mesenchymal stem cells is not particularly limited,but adipose-derived mesenchymal stem cells or bone-marrow-derivedmesenchymal stem cells are preferable, and adipose-derived mesenchymalstem cells are more preferable. A mesenchymal stem cell refers to asomatic stem cell present in the mesenchymal tissue and has an abilityto differentiate into a cell belonging to the mesenchymal tissue. Themesenchymal tissue refers to tissues such as bone, cartilage, fat,blood, bone marrow, skeletal muscle, dermis, ligament, tendon, heart,and the like.

<Cell Structure>

The cells used in the present specification may be used as they are, butthey may be used as a cell structure. The cell structure referred to inthe present invention is a cell structure which includes a plurality ofthe biocompatible polymer blocks and a plurality of cells of at leastone type and in which at least one of the biocompatible polymer blocksis disposed in gaps between the plurality of cells. In the presentspecification, a cell structure is referred to as a mosaic cell cluster(cell cluster formed in a mosaic pattern) in some cases.

(1) Biocompatible Polymer Block

(1-1) Biocompatible Polymer

Biocompatibility means that a remarkable adverse reaction such as along-term and chronic inflammatory reaction is not caused upon contactwith a living body. Regarding the biocompatible polymer used in thepresent invention, whether to be decomposed within a living body is notparticularly limited as long as the biocompatible polymer has anaffinity for the living body. However, a biodegradable polymer ispreferable. Specific examples of a non-biodegradable material includepolytetrafluoroethylene (PTFE), polyurethane, polypropylene, polyester,vinyl chloride, polycarbonate, acryl, stainless steel, titanium,silicone, and 2-methacryloyloxyethyl phosphorylcholine (MPC). Specificexamples of a biodegradable material include a polypeptide (for example,gelatin described below) such as a naturally occurring peptide, arecombinant peptide, or a chemically synthesized peptide, polylacticacid, polyglycolic acid, poly(lactic-co-glycolic acid) (PLGA),hyaluronic acid, glycosaminoglycan, proteoglycan, chondroitin,cellulose, agarose, carboxymethyl cellulose, chitin, and chitosan. Amongthese, a recombinant peptide is particularly preferable. Thebiocompatible polymers may be devised to improve cell adhesiveness.Specifically, a method such as “coating a substrate surface with celladhesion stroma (fibronectin, vitronectin, or laminin) or a peptidehaving cell adhesion sequence (an RGD sequence, an LDV sequence, an REDVsequence (SEQ ID NO: 2), a YIGSR sequence (SEQ ID NO: 3), a PDSGRsequence (SEQ ID NO: 4), an RYVVLPR sequence (SEQ ID NO: 5), an LGTIPGsequence (SEQ ID NO: 6), an RNIAEIIKDI sequence (SEQ ID NO: 7), an IKVAVsequence (SEQ ID NO: 8), an LRE sequence, a DGEA sequence (SEQ ID NO:9), and a HAV sequence, all expressed as one letter code of aminoacids)”, “amination and cationization of a substrate surface”, or “aplasma treatment and a hydrophilic treatment due to corona discharge ona substrate surface” can be used.

The type of a polypeptide including a recombinant peptide or achemically synthesized peptide is not particularly limited as long asthe polypeptide has biocompatibility. For example, gelatin, collagen,atelocollagen, elastin, fibronectin, pronectin, laminin, tenascin,fibrin, fibroin, entactin, thrombospondin, and RETRONECTIN (registeredtrademark) are preferable, and gelatin, collagen, and atelocollagen aremost preferable. Gelatin to be used in the present invention ispreferably natural gelatin, recombinant gelatin, or chemicallysynthesized gelatin, and more preferably recombinant gelatin. Thenatural gelatin referred to herein means gelatin produced usingnaturally derived collagen.

The chemically synthesized peptide and the chemically synthesizedgelatin mean an artificially synthesized peptide and artificiallysynthesized gelatin, respectively. A peptide such as gelatin may besynthesized by solid phase synthesis or liquid phase synthesis, but thesolid phase synthesis is preferable. The solid phase synthesis of thepeptide is well-known to those skilled in the art, and examples thereofinclude a fluorenyl-methoxy-carbonyl group (Fmoc group) synthesis methodin which a Fmoc group is used for protection of an amino group, and atert-butyl oxy carbonyl group (Boc group) synthesis method in which aBoc group is used for protection of an amino group. As the preferredembodiment of the chemically synthesized gelatin, the contents describedin the recombinant gelatin described below in the present specificationcan be applied.

A “1/IOB” value which is a hydrophilicity value of the biocompatiblepolymer is preferably 0 to 1.0. The 1/IOB value is more preferably 0 to0.6 and still more preferably 0 to 0.4. IOB is an index ofhydrophilicity and hydrophobicity based on an organic conceptual diagramshowing polarity and non-polarity of an organic compound, which has beenproposed by Atsushi FUJITA, and the details thereof are described in,for example, “Pharmaceutical Bulletin”, Vol. 2, 2, pp. 163 to 173(1954), “Area of Chemistry”, Vol. 11, 10, pp. 719 to 725 (1957), and“Fragrance Journal”, Vol. 50, pp. 79 to 82 (1981). Briefly, assumingthat the source of all organic compounds is methane (CH₄) and all othercompounds are derivatives of methane, predetermined numerical values areset for the number of carbon atoms, a substituent, a transformationportion, a ring, and the like of the compounds, scores thereof are addedto determine an organic value (OV) and an inorganic value (IV), andthese values are plotted on a diagram in which the organic value isplaced on an X-axis and the inorganic value is placed on a Y-axis. IOBin the organic conceptual diagram refers to a ratio of the inorganicvalue (IV) to the organic value (OV) in the organic conceptual diagram,that is, “inorganic value (IV)/organic value (OV)”. For the details ofthe organic conceptual diagram, “New Edition Organic ConceptualDiagram-Foundation and Application-” (written by Yoshio KOUDA et al.,Sankyo Shuppan Co., Ltd., 2008) can be referred to. In the presentspecification, the hydrophilicity and hydrophobicity are represented bya “1/IOB” value obtained by taking a reciprocal of IOB. A smaller“1/IOB” value (close to 0) indicates higher hydrophilicity.

It is presumed that since hydrophilicity becomes high and waterabsorbency becomes high by setting the “1/IOB” value of thebiocompatible polymer used in the present invention in the above range,the polymer effectively acts to retain nutrient components and, as aresult, contributes to the stabilization and viability of cells in thecell structure (mosaic cell cluster) according to the present invention.

In a case where the biocompatible polymer is a polypeptide, thehydrophilicity and hydrophobicity index represented by a grand averageof hydropathicity (GRAVY) value is preferably −9.0 to 0.3 and morepreferably −7.0 to 0.0. The grand average of hydropathicity (GRAVY)value can be obtained by methods of “Gasteiger E., Hoogland C., GattikerA., Duvaud S., Wilkins M. R., Appel R. D., Bairoch A.; ProteinIdentification and Analysis Tools on the ExPASy Server; (In) John M.Walker (ed): The Proteomics Protocols Handbook, Humana Press (2005). pp.571 to 607” and “Gasteiger E., Gattiker A., Hoogland C., Ivanyi I.,Appel R. D., Bairoch A.; ExPASy: the proteomics server for in-depthprotein knowledge and analysis; Nucleic Acids Res. 31:3784-3788 (2003)”.

It is presumed that since hydrophilicity becomes high and waterabsorbency becomes high by setting the GRAVY value of the biocompatiblepolymer in the above range, the polymer effectively acts to retainnutrient components and, as a result, contributes to the stabilizationand viability of cells in the cell structure (mosaic cell cluster)according to the present invention.

(1-2) Cross-Linking

The biocompatible polymers may be or may not be cross-linked, but arepreferably cross-linked. By using the cross-linked biocompatiblepolymers, it is possible to obtain an effect of preventing instantdecomposition thereof at the time of culturing in a medium and at thetime of transplantation into a living body. As general cross-linkingmethods, thermal cross-linking, cross-linking using aldehydes (forexample, formaldehyde, glutaraldehyde, or the like), cross-linking usinga condensation agent (carbodiimide, cyanamide, or the like), enzymaticcross-linking, photo cross-linking, ultraviolet cross-linking, ahydrophobic interaction, hydrogen bonding, an ionic interaction, and thelike are known, and the cross-linking methods can be used in the presentinvention. As the cross-linking methods used in the present invention,thermal cross-linking, ultraviolet cross-linking, or enzymaticcross-linking is more preferable, and thermal cross-linking isparticularly preferable.

In a case of performing cross-linking using an enzyme, the enzyme is notparticularly limited as long as the enzyme has a function ofcross-linking polymer materials. However, it is possible to performcross-linking preferably using transglutaminase and laccase, and mostpreferably using transglutaminase. Specific examples of proteins whichare enzymatically cross-linked by transglutaminase are not particularlylimited as long as the proteins have a lysine residue and a glutamineresidue. Transglutaminase may be derived from a mammal or amicroorganism. Specific examples thereof include ACTIVA seriesmanufactured by Ajinomoto Co., Inc., mammal-derived transglutaminasewhich is sold as a reagent, for example, guinea pig liver-derivedtransglutaminase, goat-derived transglutaminase, and rabbit-derivedtransglutaminase, which are manufactured by Oriental Yeast Co., Ltd.,Upstate USA Inc., and Biodesign International Inc., and human-derivedblood coagulation factor (Factor XIIIa, Haematologic Technologies,Inc.).

The reaction temperature in a case of performing cross-linking (forexample, thermal cross-linking) is not particularly limited as long ascross-linking can be performed, but is preferably −100° C. to 500° C.,more preferably 0° C. to 300° C., still more preferably 50° C. to 300°C., particularly preferably 100° C. to 250° C., and most preferably 120°C. to 200° C.

(1-3) Recombinant Gelatin

The recombinant gelatin referred to in the present invention means apolypeptide or a protein-like substance which is produced by generecombination technology and has an amino acid sequence similar to thatof gelatin. The recombinant gelatin which can be used in the presentinvention preferably has repetition of a sequence represented by Gly-X-Y(X and Y each independently represent any amino acid) which ischaracteristic of collagen. Herein, a plurality of pieces of Gly-X-Y maybe the same as or different from each other. Preferably, two or moresequences of cell adhesion signals are included in one molecule. As therecombinant gelatin used in the present invention, it is possible to userecombinant gelatin having an amino acid sequence derived from a partialamino acid sequence of collagen. For example, it is possible to userecombinant gelatin disclosed in EP1014176, U.S. Pat. No. 6,992,172B,WO2004/085473A, and WO2008/103041A, but the present invention is notlimited thereto. A preferred example of the recombinant gelatin used inthe present invention is recombinant gelatin of the following aspect.

The recombinant gelatin is excellent in biocompatibility due to originalcharacteristics of natural gelatin, is not naturally derived so thatthere is no concern about bovine spongiform encephalopathy (BSE) or thelike, and is excellent in non-infection properties. The recombinantgelatin is more uniform than natural gelatin, and a sequence thereof isdetermined. Accordingly, it is possible to precisely design the strengthand degradability with less fluctuation due to cross-linking or thelike.

The molecular weight of the recombinant gelatin is not particularlylimited, and is preferably 2,000 to 100,000 (2 kilodaltons (kDa) to 100kDa), more preferably 2,500 to 95,000 (2.5 kDa to 95 kDa), still morepreferably 5,000 to 90,000 (5 kDa to 90 kDa), and most preferably 10,000to 90,000 (10 kDa to 90 kDa).

The recombinant gelatin preferably has repetition of a sequencerepresented by Gly-X-Y which is characteristic of collagen. Herein, aplurality of pieces of Gly-X-Y may be the same as or different from eachother. In Gly-X-Y, Gly represents glycine and X and Y represent anyamino acid (preferably represents any amino acid other than glycine).The sequence represented by Gly-X-Y which is characteristic of collagenis a partial structure which is extremely specific in a sequence and acomposition of an amino acid of gelatin and collagen, compared withother proteins. In this portion, glycine occupies about one third of theentire sequence, and is repeated at every third position in an aminoacid sequence. Glycine is the simplest amino acid, has little restrainton the arrangement of the molecular chains, and significantlycontributes to regeneration of a helix structure during gelation. It ispreferable that amino acids represented by X and Y contain many iminoacids (proline and oxyproline) and occupy 10% to 45% of the entiresequence. Preferably 80% or more, more preferably 95% or more, and mostpreferably 99% or more of the amino acids in the sequence of therecombinant gelatin have a repeating structure of Gly-X-Y.

In general gelatin, a polar charged amino acid and a polar unchargedamino acid are present at a ratio of 1:1. Herein, the polar amino acidspecifically indicates cysteine, aspartic acid, glutamic acid,histidine, lysine, asparagine, glutamine, serine, threonine, tyrosine,and arginine. Among these, the polar uncharged amino acid indicatescysteine, asparagine, glutamine, serine, threonine, and tyrosine. In therecombinant gelatin used in the present invention, the proportion of thepolar amino acid in the whole constituent amino acid is 10% to 40% andis preferably 20% to 30%. The proportion of the uncharged amino acid inthe polar amino acids is greater than or equal to 5% and less than 20%and is preferably greater than or equal to 5% and less than 10%. It ispreferable that any one amino acid or preferably two or more amino acidsamong serine, threonine, asparagine, tyrosine, and cysteine are notcontained on the sequence.

In general, a minimum amino acid sequence which functions as a celladhesion signal in a polypeptide is known (for example, Nagai ShotenCo., Ltd., “Pathophysiology”, Vol. 9, No. 7 (1990), p. 527). Therecombinant gelatin used in the present invention preferably has two ormore cell adhesion signals in one molecule. As the specific sequence,sequences such as an RGD sequence, an LDV sequence, an REDV sequence(SEQ ID NO: 2), a YIGSR sequence (SEQ ID NO: 3), a PDSGR sequence (SEQID NO: 4), an RYVVLPR sequence (SEQ ID NO: 5), an LGTIPG sequence (SEQID NO: 6), an RNIAEIIKDI sequence (SEQ ID NO: 7), an IKVAV sequence (SEQID NO: 8), an LRE sequence, a DGEA sequence (SEQ ID NO: 9), and a HAVsequence, which are expressed as one letter code of amino acids, arepreferable because many kinds of cells adhere to these sequences. An RGDsequence, a YIGSR sequence (SEQ ID NO: 3), a PDSGR sequence (SEQ ID NO:4), an LGTIPG sequence (SEQ ID NO: 6), an IKVAV sequence (SEQ ID NO: 8),and a HAV sequence are more preferable, and an RGD sequence isparticularly preferable. In the RGD sequences, an ERGD sequence (SEQ IDNO: 10) is preferable. A production amount of stroma of a cell can beimproved by using recombinant gelatin having cell adhesion signals.

As the disposition of RGD sequences in the recombinant gelatin, it ispreferable that the number of amino acids between RGDs is not uniformbetween 0 and 100, and it is more preferable that the number of aminoacids between RGDs is not uniform between 25 and 60. The content of thisminimum amino acid sequence unit is preferably 3 to 50, more preferably4 to 30, particularly preferably 5 to 20, and most preferably 12 in onemolecule of a protein in the viewpoint of cell adhesion andproliferation properties.

In the recombinant gelatin, a proportion of an RGD motif with respect tothe total number of amino acids is preferably at least 0.4%. In a casewhere the recombinant gelatin contains 350 or more amino acids, eachstretch of the 350 amino acids preferably contains at least one RGDmotif. The proportion of the RGD motif with respect to the total numberof amino acids is more preferably at least 0.6%, still more preferablyat least 0.8%, even more preferably at least 1.0%, particularlypreferably at least 1.2%, and most preferably at least 1.5%. The numberof RGD motifs within a recombinant peptide is preferably at least 4,more preferably at least 6, still more preferably at least 8, andparticularly preferably 12 to 16 per 250 amino acids. The proportion ofthe RGD motif of 0.4% corresponds to at least one RGD sequence per 250amino acids. The number of RGD motifs is an integer, and accordingly,gelatin consisting of 251 amino acids needs to contain at least two RGDsequences in order to satisfy the characteristics of 0.4%. Therecombinant gelatin preferably contains at least two RGD sequences per250 amino acids, more preferably contains at least three RGD sequencesper 250 amino acids, and still more preferably contains at least fourRGD sequences per 250 amino acids. As another aspect of the recombinantgelatin, the recombinant gelatin preferably contains at least 4 RGDmotifs, more preferably contains at least 6 RGD motifs, still morepreferably contains at least 8 RGD motifs, and particularly preferablycontains 12 to 16 RGD motifs.

The recombinant gelatin may be partially hydrolyzed.

The recombinant gelatin is preferably represented by Formula 1:A-[(Gly-X-Y)_(n)]_(m)-B. n pieces of X each independently represent anyamino acid and n pieces of Y each independently represent any aminoacid. m preferably represents an integer of 2 to 10 and more preferablyrepresents an integer of 3 to 5. n is preferably an integer of 3 to 100,more preferably an integer of 15 to 70, and most preferably an integerof 50 to 65. A represents any amino acid or any amino acid sequence andB represents any amino acid or any amino acid sequence. n pieces ofGly-X-Y may be the same as or different from each other.

More preferably, the recombinant gelatin is represented byGly-Ala-Pro-[(Gly-X-Y)₆₃]₃-Gly (SEQ ID NO: 11)(in the formula, 63 piecesof X each independently represent any amino acid and 63 pieces of Y eachindependently represent any amino acid. 63 pieces of Gly-X-Y may be thesame as or different from each other).

It is preferable that a plurality of sequence units of naturallyexisting collagen are bonded to a repeating unit. The naturally existingcollagen referred to herein is not limited as long as the collagenexists naturally, but is preferably I type collagen, II type collagen,III type collagen, IV type collagen, or V type collagen, and morepreferably I type collagen, II type collagen, or III type collagen.According to another embodiment, the above-described collagen is derivedpreferably from a human, cattle, a pig, a mouse, or a rat, and morepreferably from a human.

An isoelectric point of the recombinant gelatin is preferably 5 to 10,more preferably 6 to 10, and still more preferably 7 to 9.5. Themeasurement of the isoelectric point of the recombinant gelatin can becarried out by measuring a pH after passing a 1 mass % gelatin solutionthrough a mixed crystal column of a cation-anion exchange resin, asdescribed in the isoelectric focusing method (refer to Maxey, C. R.(1976); Phitogr. Gelatin 2, Editor Cox, P. J. Academic, London, Engl.).

It is preferable that the recombinant gelatin is not deaminated.

It is preferable that the recombinant gelatin does not have atelopeptide.

It is preferable that the recombinant gelatin is a substantially purepolypeptide prepared using a nucleic acid which encodes an amino acidsequence.

It is particularly preferable that the recombinant gelatin is any of

(1) a peptide consisting of an amino acid sequence described in SEQ IDNO: 1;

(2) a peptide which is formed of an amino acid sequence in which one orseveral amino acids are deleted, substituted, or added in the amino acidsequence described in SEQ ID No: 1, and has biocompatibility; or

(3) a peptide which is formed of an amino acid sequence having 80% ormore (more preferably 90% or more, particularly preferably 95% or more,and most preferably 98% or more) sequence identity to the amino acidsequence described in SEQ ID No: 1, and has biocompatibility.

The sequence identity in the present invention refers to a valuecalculated by the following expression.% Sequence identity=[(the number of identical residues)/(alignmentlength)]×100

The sequence identity between two amino acid sequences can be determinedby any method well-known to those skilled in the art, and can bedetermined by using a basic local alignment search tool (BLAST) program(J. Mol. Biol. 215: 403 to 410, 1990) or the like.

“One or several” in the expression “amino acid sequence in which one orseveral amino acids are deleted, substituted, or added” means preferably1 to 20 amino acids, more preferably 1 to 10 amino acids, still morepreferably 1 to 5 amino acids, and particularly preferably 1 to 3 aminoacids.

The recombinant gelatin can be produced by a gene recombinationtechnology which is well-known to those skilled in the art, and can beproduced, for example, in accordance with methods disclosed inEP1014176A2, U.S. Pat. No. 6,992,172B, WO2004/085473A, andWO2008/103041A. Specifically, a gene encoding an amino acid sequence ofpredetermined recombinant gelatin is acquired, the acquired gene isincorporated into an expression vector to produce a recombinantexpression vector, and a transformant is produced by introducing therecombinant expression vector into an appropriate host. The recombinantgelatin is produced by culturing the obtained transformant in anappropriate medium. Accordingly, the recombinant gelatin used in thepresent invention can be prepared by collecting the recombinant gelatinproduced from a culture product.

(1-4) Biocompatible Polymer Block

In the present invention, a block (cluster) formed of theabove-described biocompatible polymers can be used.

The shape of the biocompatible polymer block in the present invention isnot particularly limited. Examples thereof include an amorphous shape, aspherical shape, a particulate shape (granule), a powdery shape, aporous shape, a fibrous shape, a spindle shape, a flat shape, and asheet shape, and an amorphous shape, a spherical shape, a particulateshape (granule), a powdery shape, and a porous shape are preferable. Theamorphous shape indicates that a shape of a surface is uneven, andindicates, for example, an object having roughness, such as rock.Examples of the above-described shapes are not distinct from each other,and, for example, an amorphous shape is included in an example of asubordinate concept of the particulate shape (granule) in some cases.

The shape of the biocompatible polymer block in the present invention isnot particularly limited as described above. However, tap density ispreferably 10 mg/cm³ to 500 mg/cm³, more preferably 20 mg/cm³ to 400mg/cm³, still more preferably 40 mg/cm³ to 220 mg/cm³, and particularlypreferably 50 mg/cm³ to 150 mg/cm³.

The tap density is a value indicating how many blocks can be denselypacked in a certain volume, and it is apparent that as the value becomeslower, the blocks cannot be densely packed, that is, the structure ofthe block is complicated. It is considered that the tap density of thebiocompatible polymer block indicates complexity of a surface structureof the biocompatible polymer block and a volume of a void formed in acase where biocompatible polymer blocks are collected as an aggregate.As the tap density becomes smaller, the void between biocompatiblepolymer blocks becomes larger and a grafted region of a cell becomeslarger. In addition, by setting the tap density to be not too small, thebiocompatible polymer block can appropriately exist between cells, andin a case where a cell structure is formed, nutrients can be deliveredinto the cell structure. Therefore, it is considered to be preferablethat the tap density falls within the above range.

The measurement of the tap density referred to in the presentspecification is not particularly limited, but can be performed asfollows. A container (having a cylindrical shape with a diameter of 6 mmand a length of 21.8 mm: a capacity of 0.616 cm³) (hereinafter,described as a cap) is prepared for the measurement. First, a mass ofonly the cap is measured. Then, a funnel is attached to the cap, andblocks are poured from the funnel so as to be accumulated in the cap.After pouring a sufficient amount of blocks, the cap portion is hit 200times on a hard object such as a desk, the funnel is removed, and theblocks are leveled off with a spatula. A mass is measured in a statewhere the cap is filled up with the blocks. The tap density can bedetermined by calculating a mass of only the blocks from the differencebetween the mass of the cap filled up with the blocks and the mass ofonly the cap, and dividing the mass of only the blocks by the volume ofthe cap.

A cross-linking degree of the biocompatible polymer block in the presentinvention is not particularly limited, but is preferably greater than orequal to 2, more preferably 2 to 30, still more preferably 4 to 25, andparticularly preferably 4 to 22.

The method of measuring the cross-linking degree (the number of times ofcross-linking per molecule) of the biocompatible polymer block is notparticularly limited. However, in a case where the biocompatible polymeris CBE3, the measurement can be performed, for example, by a TNBS(2,4,6-trinitrobenzene sulfonic acid) method described in the followingexamples. Specifically, a sample obtained by mixing a biocompatiblepolymer block, a NaHCO₃ aqueous solution, and a TNBS aqueous solution,allowing the mixture to react for 3 hours at 37° C., and then stoppingthe reaction, and a blank obtained by mixing a biocompatible polymerblock, a NaHCO₃ aqueous solution, and a TNBS aqueous solution andstopping a reaction immediately after the mixing are prepared. Eachabsorbance (345 nm) of the sample and the blank which are diluted withpure water is measured, and the cross-linking degree (the number oftimes of cross-linking per molecule) can be calculated from (Expression2) and (Expression 3).(As−Ab)/14,600×V/w  (Expression 2)

(Expression 2) represents an amount (molar equivalent) of lysine per 1 gof the biocompatible polymer block.

(In the expression, As represents a sample absorbance, Ab represents ablank absorbance, V represents an amount (g) of reaction liquid, and wrepresents a mass (mg) of the biocompatible polymer block.)1−(sample (Expression 2)/uncross-linked polymer (Expression2))×34  (Expression 3)

(Expression 3) represents the number of times of cross-linking permolecule.

A water absorption rate of the biocompatible polymer block in thepresent invention is not particularly limited, but is preferably greaterthan or equal to 300%, more preferably greater than or equal to 400%,still more preferably greater than or equal to 500%, particularlypreferably greater than or equal to 600%, and most preferably greaterthan or equal to 700%. An upper limit of the water absorption rate isnot particularly limited, but is generally less than or equal to 4,000%or less than or equal to 2,000%.

The method of measuring the water absorption rate of the biocompatiblepolymer block is not particularly limited. However, the water absorptionrate can be measured, for example, by the method described in thefollowing examples. Specifically, a 3 cm×3 cm bag made of nylon mesh isfilled with about 15 mg of a biocompatible polymer block at 25° C., isswollen in ion exchange water for 2 hours, and then is dried with airfor 10 minutes, the mass thereof is measured at each stage, the waterabsorption rate is determined according to (Expression 4).Water absorption rate=(w2−w1−w0)/w0  (Expression 4)

(In the expression, w0 represents a mass of a material before waterabsorption, w1 represents a mass of an empty bag after water absorption,and w2 represents a mass of the whole bag containing the material afterwater absorption.)

The size of one biocompatible polymer block in the present invention isnot particularly limited, but is preferably 20 μm to 200 μm, morepreferably 20 μm to 150 μm, still more preferably 50 μm to 120 μm, andparticularly preferably 53 μm to 106 μm.

By setting the size of one biocompatible polymer block in the aboverange, nutrient delivery into a cell structure from the outside can beimproved. The size of one biocompatible polymer block does not mean thatan average value of the sizes of a plurality of biocompatible polymerblocks is within the above range, but means the size of eachbiocompatible polymer block which is obtained by sieving a plurality ofbiocompatible polymer blocks.

The size of one block can be defined by a size of a sieve used in a caseof dividing the blocks. For example, blocks remaining on a sieve with106 μm in a case where blocks which have been passed through a sievewith 180 μm for sifting are sifted using the sieve with 106 μm can beregarded as blocks having a size of 106 to 180 μm. Next, blocksremaining on a sieve with 53 μm in a case where blocks which have beenpassed through the sieve with 106 μm for sifting are sifted using thesieve with 53 μm can be regarded as blocks having a size of 53 to 106μm. Next, blocks remaining on a sieve with 25 μm in a case where blockswhich have been passed through the sieve with 53 μm for sifting aresifted using the sieve with 25 μm can be regarded as blocks having asize of 25 to 53 μm.

(1-5) Method of Producing Biocompatible Polymer Block

The method of producing a biocompatible polymer block is notparticularly limited. For example, it is possible to obtain abiocompatible polymer block by pulverizing a solid matter (such as aporous body of a biocompatible polymer) containing a biocompatiblepolymer using a pulverizer (such as NEW POWER MILL). The solid matter(such as a porous body) containing a biocompatible polymer can beobtained, for example, by freeze-drying an aqueous solution containingthe biocompatible polymer.

By pulverizing the solid matter containing a biocompatible polymer asdescribed above, an amorphous biocompatible polymer block having anuneven surface shape can be produced.

The method of producing the porous body of the biocompatible polymer isnot particularly limited, but the porous body can also be obtained byfreeze-drying an aqueous solution containing a biocompatible polymer.For example, by including a freezing step in which the liquidtemperature (highest internal highest liquid temperature) of a portionhaving the highest liquid temperature in the solution is lower than orequal to “solvent melting point−3° C.” in the unfrozen state, the ice tobe formed can have a spherical shape. By drying the ice after performingthis step, a porous body having spherical isotropic holes (sphericalpores) can be obtained. For example, by performing freezing withoutincluding a freezing step in which the liquid temperature (highestinternal liquid temperature) of a portion having the highest liquidtemperature in the solution is higher than or equal to “solvent meltingpoint−3° C.” in the unfrozen state, the ice to be formed can have apillar/flat plate shape. By drying the ice after performing this step, aporous body having holes (pillars/flat plate pores) with pillar or flatshapes which are long uniaxially or biaxially can be obtained. In a casewhere the porous body of the biocompatible polymer is pulverized toproduce a biocompatible polymer block, the holes of the porous bodybefore pulverization influence the shape of the biocompatible polymerblock to be obtained, and thus the shape of the biocompatible polymerblock to be obtained can be adjusted by adjusting the condition offreeze-drying as described above.

An example of a method of producing a porous body of a biocompatiblepolymer includes a method including

a step (a) of cooling a solution of biocompatible polymers to anunfrozen state under the conditions where the difference between atemperature of a portion having the highest liquid temperature in thesolution and a temperature of a portion having the lowest liquidtemperature in the solution is lower than or equal to 2.5° C. and thetemperature of the portion having the highest liquid temperature in thesolution is lower than or equal to a melting point of a solvent;

a step (b) of freezing the solution of the biocompatible polymersobtained in the step (a); and

a step (c) of freeze-drying the frozen biocompatible polymers obtainedin the step (b).

However, the present invention is not limited to the above method.

In a case where the solution of the biocompatible polymers is cooled toan unfrozen state, the variation in the sizes of obtained porous poresis reduced by making the difference between the temperature of theportion having the highest liquid temperature and the temperature of theportion having the lowest liquid temperature in the solution be lowerthan or equal to 2.5° C. (preferably lower than or equal to 2.3° C. andmore preferably lower than or equal to 2.1° C.), that is, by reducingthe difference in temperature. A lower limit of the difference betweenthe temperature of the portion having the highest liquid temperature andthe temperature of the portion having the lowest liquid temperature inthe solution is not particularly limited, but may be higher than orequal to 0° C. For example, the lower limit thereof may be higher thanor equal to 0.1° C., higher than or equal to 0.5° C., higher than orequal to 0.8° C., or higher than or equal to 0.9° C. Accordingly, thecell structure using the biocompatible polymer block which is producedwith the produced porous body achieves the effect of showing a largenumber of cells.

The cooling in the step (a) is preferably carried out, for example,using a material (preferably TEFLON (registered trademark)) having alower thermal conductivity than water. The portion having the highestliquid temperature in the solution can be supposed as the farthestportion from a cooling side, and the portion having the lowest liquidtemperature in the solution can be supposed as a liquid temperature ofthe cooled surface.

In the step (a), the difference between the temperature of the portionhaving the highest liquid temperature in the solution and thetemperature of the portion having the lowest liquid temperature in thesolution, immediately before generation of solidification heat, ispreferably lower than or equal to 2.5° C., more preferably lower than orequal to 2.3° C., and still more preferably lower than or equal to 2.1°C. Here, the “difference in temperature immediately before thegeneration of solidification heat” means a difference in temperature ina case where the difference in temperature is the largest between 1second and 10 seconds before the generation of solidification heat.

In the step (a), the temperature of the portion having the lowest liquidtemperature in the solution is preferably lower than or equal to“solvent melting point−5° C.”, more preferably lower than or equal to“solvent melting point−5° C.” and higher than or equal to “solventmelting point−20° C.”, and still more preferably lower than or equal to“solvent melting point−6° C.” and higher than or equal to “solventmelting point−16° C.”. The solvent in the solvent melting point refersto a solvent of a solution of biocompatible polymers.

In the step (b), the solution of the biocompatible polymers obtained inthe step (a) is frozen. The cooling temperature for the freezing in thestep (b) is not particularly limited and depends on cooling equipment.However, the cooling temperature is a temperature which is lower thanthe temperature of the portion having the lowest liquid temperature inthe solution preferably by 3° C. to 30° C., more preferably by 5° C. to25° C., and still more preferably by 10° C. to 20° C.

In the step (c), the frozen biocompatible polymers obtained in the step(b) are freeze-dried. The freeze-drying can be performed by a usualmethod. For example, the freeze-drying can be performed by carrying outvacuum drying at a temperature lower than a melting point of a solventand further carrying out vacuum drying at room temperature (20° C.).

In the present invention, a biocompatible polymer block can be producedpreferably by pulverizing the porous body obtained in theabove-described step (c).

(2) Cell Structure

The cell structure is a cell structure which includes a plurality of thebiocompatible polymer blocks and a plurality of cells of at least onetype and in which at least one of the polymer blocks is disposed in gapsbetween the plurality of cells. The biocompatible polymer blocks and thecells are used and the plurality of polymer blocks arethree-dimensionally disposed in a mosaic pattern in the gaps between theplurality of cells. By three-dimensionally disposing the biocompatiblepolymer blocks and the cells in a mosaic pattern, a three-dimensionalcell structure in which cells uniformly exist in the structure is formedand material permeability is obtained.

In the cell structure, the plurality of polymer blocks are disposed ingaps between the plurality of cells. Here, the “gaps between cells” arenot necessarily spaces closed by the constituent cells, and may beinterposed between the cells. Moreover, gaps are not necessarily presentbetween all of the cells, and there may be a place where the cells arein contact with each other. The distance of a gap between cells throughthe polymer block, that is, the gap distance in a case of selecting acertain cell and a cell present at the shortest distance from thecertain cell is not particularly limited. However, the distance ispreferably the same as the size of a polymer block, and a suitabledistance is also within a range of a suitable size of a polymer block.

Furthermore, the polymer blocks are configured to be interposed betweenthe cells. However, cells are not necessarily present between all of thepolymer blocks, and there may be a place where the polymer blocks are incontact with each other. The distance between polymer blocks through thecell, that is, the distance in a case of selecting a polymer block and apolymer block present at the shortest distance from the polymer block isnot particularly limited. However, the distance is preferably the sameas a size of a cluster of cells in a case where one or several cells tobe used are gathered and for example, the size thereof is 10 μm to 1,000μm, preferably 10 μm to 100 μm, and more preferably 10 μm to 50 μm.

In the present specification, the expression “uniformly exist” in“three-dimensional cell structure in which cells uniformly exist in thestructure” or the like is used, but does not mean complete uniformity.

A thickness or a diameter of the cell structure can be set to a desiredsize, but a lower limit thereof is preferably greater than or equal to100 μm, more preferably greater than or equal to 215 μm, still morepreferably greater than or equal to 400 μm, and most preferably greaterthan or equal to 730 μm. An upper limit of the thickness or the diameteris not particularly limited, but a general range thereof in use ispreferably less than or equal to 3 cm, more preferably less than orequal to 2 cm, and still more preferably less than or equal to 1 cm. Therange of the thickness or the diameter of the cell structure ispreferably 100 μm to 3 cm, more preferably 400 μm to 3 cm, still morepreferably 500 μm to 2 cm, and even more preferably 720 μm to 1 cm.

In the cell structure, regions including polymer blocks and regionsincluding cells are preferably disposed in a mosaic pattern. Moreover,in the present specification, the expression “the thickness or thediameter of the cell structure” means the followings. In a case whereone point A in the cell structure is selected, a length of a linesegment which divides the cell structure so that the distance from anouter boundary of the cell structure is the shortest in a straight linepassing through the point A is set as a line segment A. The point A atwhich the line segment A becomes the longest is selected in the cellstructure, and a length of the line segment A in this case is set as “athickness or a diameter of a cell structure”.

In the cell structure, a ratio of a polymer block to a cell is notparticularly limited. However, a mass of a polymer block per cell ispreferably 0.0000001 μg to 1 μg, more preferably 0.000001 μg to 0.1 μg,still more preferably 0.00001 μg to 0.01 μg, and most preferably 0.00002μg to 0.006 μg. By setting the ratio in the above range, the cells canfurther uniformly exist. By setting a lower limit thereof in the aboverange, the effect of the cell can be exhibited in case of using the cellstructure for the above-described application, and by setting an upperlimit thereof in the above range, components optionally present in thepolymer block can be supplied to the cell. Here, the components in thepolymer block are not particularly limited, but examples thereof includecomponents contained in a medium described below.

(3) Method of Producing Cell Structure

The cell structure can be produced by mixing biocompatible polymerblocks and at least one type of cells. Specifically, the cell structurecan be produced by alternately disposing the biocompatible polymerblocks (cluster including biocompatible polymers) and the cells.Moreover, “alternately” does not mean complete alternation, but forexample, means a state where the biocompatible polymer blocks and thecells are mixed. The production method is not particularly limited, butis preferably a method of seeding cells after a polymer block is formed.Specifically, a cell structure can be produced by incubating a mixtureof the biocompatible polymer blocks and a cell-containing culturesolution. For example, the cells and the biocompatible polymer blocksproduced in advance are disposed in a mosaic pattern in a container orin a liquid held in a container. As a means of disposition, it ispreferable to promote and control formation of mosaic-like dispositionincluding the cells and the biocompatible polymer blocks by usingspontaneous aggregation, natural falling, centrifugation, and stirring.

The container to be used is preferably a container made of a celllow-adhesive material or a cell non-adhesive material, and morepreferably a container made of polystyrene, polypropylene, polyethylene,glass, polycarbonate, and polyethylene terephthalate. A shape of abottom surface of the container is preferably a flat bottom shape, a Ushape, or a V shape.

With respect to the mosaic-patterned cell structure obtained by theabove method, a cell structure having a desired size can be produced bya method such as (a) fusing mosaic cell clusters which are separatelyprepared, or (b) increasing a volume thereof in a differentiation mediumor a proliferation medium. The method of fusion and the method ofincreasing a volume are not particularly limited.

For example, in a step of incubating the mixture of the biocompatiblepolymer blocks and the cell-containing culture solution, the volume ofthe cell structure can be increased by replacing the medium with adifferentiation medium or a proliferation medium. Preferably, in thestep of incubating the mixture of the biocompatible polymer blocks andthe cell-containing culture solution, a cell structure which has adesired size and in which cells uniformly exist can be produced byfurther adding the biocompatible polymer blocks.

Specifically, the method of fusing mosaic cell clusters which areseparately prepared is a method of producing a cell structure includinga step of fusing a plurality of cell structures which include aplurality of biocompatible polymer blocks and a plurality of cells andin which one or the plurality of biocompatible polymer blocks aredisposed in a part or all of a plurality of gaps formed by the pluralityof cells.

<Immunoisolation Membrane>

In the present specification, an immunoisolation membrane means amembrane used for immunoisolation.

Immunoisolation is a method of preventing immune rejection. In general,immunoisolation is one method of preventing immune rejection of arecipient during transplantation. The immune rejection is the rejectionof a recipient to a cell structure to be transplanted. A cell structureis sequestered from immune rejection of a recipient. Examples of immunerejection includes immune rejection occurring due to cellular immuneresponses, and immune rejection occurring due to humoral immuneresponses.

The immunoisolation membrane is a selectively permeable membrane thatallows nutrients such as oxygen, water, and glucose to permeatetherethrough, and inhibits permeation of immune cells and the likeinvolved in an immune rejection. Examples of immune cells includemacrophages, dendritic cells, neutrophils, eosinophils, basophils,natural killer cells, various T cells, B cells, and other lymphocytes.

Depending on the application, the immunoisolation membrane preferablyinhibits permeation of high-molecular-weight proteins such asimmunoglobulins (IgM, IgG, and the like) and complements, and preferablyallows a relatively low-molecular-weight physiologically activesubstances such as insulin to permeate therethrough.

The selective permeability of the immunoisolation membrane may beadjusted according to the application. The immunoisolation membrane maybe a selectively permeable membrane which blocks a substance having amolecular weight such as 500 kDa or more, 100 kDa or more, 80 kDa ormore, or 50 kDa or more. For example, it is preferable that theimmunoisolation membrane be capable of inhibiting permeation of thesmallest IgG (molecular weight of about 160 kDa) among antibodies. Inaddition, the immunoisolation membrane may be a selectively permeablemembrane which blocks a substance having a diameter such as 500 nm ormore, 100 nm or more, 50 nm or more, or 10 nm or more, as a sphere size.

The immunoisolation membrane preferably includes a porous membranecontaining a polymer. The immunoisolation membrane may consist only of aporous membrane containing a polymer, or may include other layers.Examples of other layers include a hydrogel film. The immunoisolationmembrane may have a protective film that can be easily peeled off from asurface for transportation or the like.

A thickness of the immunoisolation membrane is not particularly limited.It is sufficient for the thickness thereof to be 10 μm to 500 μm. Thethickness thereof is preferably 20 μm to 300 μm, and is more preferably30 μm to 250 μm.

[Porous Membrane]

(Structure of Porous Membrane)

The porous membrane is a membrane having a plurality of pores. Pores canbe checked by, for example, a scanning electron microscope (SEM) imageof a cross section of the membrane, or a transmission electronmicroscope (TEM) image thereof.

A thickness of the porous membrane is not particularly limited, but itis preferably 10 μm to 500 μm, is more preferably 10 μm to 300 μm, andis even more preferably 10 μm to 250 μm.

Preferably, within an inner side of the porous membrane, a layeredcompact portion in which a pore diameter is minimized is present, and apore diameter continuously increases in a thickness direction from thecompact portion toward at least one surface of the porous membrane. Apore diameter is determined based on an average pore diameter ofsections to be described later.

A surface of the membrane means a main surface (a front surface or aback surface indicating an area of the membrane), and does not mean asurface in a thickness direction at the end of the membrane. The surfaceof the porous membrane may be an interface with another layer. In theimmunoisolation membrane, the porous membrane preferably has a uniformstructure over the entire area with respect to a pore diameter or porediameter distribution (a difference in pore diameter in the thicknessdirection), and the like.

The porous membrane having a pore diameter distribution can prolong thelife of the immunoisolation membrane. The reason for this is because ofan effect as if multi-stage filtration has been performed usingmembranes having substantially different pore diameters, and therebydeterioration of the membrane can be prevented.

A pore diameter may be measured from a photograph of a cross section ofthe membrane obtained by an electron microscope. The porous membrane canbe cut with a microtome or the like, and it is possible to obtain aphotograph of a cross section of the porous membrane as a section of athin membrane which a cross section can be observed.

In the present specification, comparison of the pore diameter in thethickness direction of the membrane is performed by dividing the SEMphotograph of the cross section of the membrane in the thicknessdirection of the membrane. The number of divisions can be appropriatelyselected according to a thickness of the membrane. The number ofdivisions is at least 5 or more. For example, in a case of a membranehaving a thickness of 200 μm, division is performed 20 times from asurface X to be described later. A size of a division width means awidth in the thickness direction of the membrane, and does not mean awidth in the photograph. In comparison of a pore diameter in thethickness direction of the membrane, the pore diameter is compared as anaverage pore diameter of each section. An average pore diameter of eachsection may be, for example, an average value of 50 pores in eachsection of a membrane cross-sectional view. The membrane cross-sectionalview in this case may be obtained, for example, with a width of 80 μm (adistance of 80 μm in a direction parallel to a surface). In this case,with respect to a section in which 50 pores cannot be measured becausepores are large, it is sufficient to measure as many as the number thatcan be taken in the section. In addition, in this case, in a case wherea pore is large and does not fit within the section, a size of the poreis measured over the other sections.

The layered compact portion having the smallest pore diameter refers toa layered portion of the porous membrane corresponding to a sectionwhere an average pore diameter becomes smallest among sections of themembrane cross section. The compact portion may consist of portionscorresponding to one section, or may consist of portions correspondingto a plurality of sections such as 2 or 3 sections, which have anaverage pore diameter within 1.1 times that of a section where anaverage pore diameter is minimum. It is sufficient for a thickness ofthe compact portion to be 0.5 μm to 50 μm, and it is preferably 0.5 μmto 30 μm. In the present specification, an average pore diameter of thecompact portion is denoted as the minimum pore diameter of the porousmembrane. The minimum pore diameter of the porous membrane is preferably0.02 μm to 1.5 μm, and is more preferably 0.02 μm to 1.3 μm. The reasonfor this is because such a minimum pore diameter of the porous membranecan prevent permeation of at least normal cells. Here, an average porediameter of the compact portion is measured by ASTM F316-80.

The porous membrane has the compact portion within an inner sidethereof. The inner side means that the compact portion is not in contactwith the surface of the membrane. The phrase “having the compact portionwithin the inner side thereof” means that the compact portion is not theclosest section to any surface of the membrane. In the immunoisolationmembrane, by using the porous membrane having a structure having thecompact portion within the inner side thereof, permeability of asubstance intended to permeate therethrough is unlikely to be diminishedas compared to a case of using a porous membrane having the same compactportion in contact with the surface thereof. Although not bound by anytheory, it is perceived that protein adsorption is less likely to occurdue to the presence of the compact portion within the inner side.

It is preferable that the compact portion be biased to one of the frontsurface side than a central portion in thickness of the porous membrane.Specifically, the compact portion is preferably located between any onesurface of the porous membrane and a portion at a distance of two-fifthof the porous membrane from the surface, is more preferably locatedbetween any one surface of the porous membrane and a portion at adistance of one-third of the porous membrane from the surface, and iseven more preferably located between any one surface of the porousmembrane and a portion at a distance of one-fourth of the porousmembrane from the surface. This distance may be determined from thephotograph of the membrane cross section described above. In the presentspecification, the surface of the porous membrane closer to the compactportion is referred to as a “surface X”.

In the porous membrane, a pore diameter continuously increases in thethickness direction from the compact portion toward at least one of thesurfaces. In the porous membrane, the pore diameter may continuouslyincrease in the thickness direction toward the surface X from thecompact portion, the pore diameter may continuously increase in thethickness direction toward the surface opposite to the surface X fromthe compact portion, and the pore diameter may continuously increase inthe thickness direction toward any surface of the porous membrane fromthe compact portion. Among them, it is preferable that the pore diametercontinuously increase in the thickness direction toward at least thesurface opposite to the surface X from the compact portion, and it ispreferable that the pore diameter continuously increase in the thicknessdirection toward any surface of the porous membrane from the compactportion. The sentence “the pore diameter continuously increases in thethickness direction” means that a difference in average pore diametersbetween sections adjacent to each other in the thickness directionincreases by 50% or less of a difference between maximum average porediameters (maximum pore diameter) and minimum average pore diameters(minimum pore diameter), preferably increases by 40% or less, and morepreferably increases by 30% or less. The phrase “continuouslyincreasing” essentially means that a pore diameter increases uniformlywithout decreasing, but a decreasing portion may occur accidentally. Forexample, in a case of combining two sections from the surface, in a casewhere an average value of a combination increases uniformly (uniformlydecreases toward the compact portion from the surface), it can bedetermined that “the pore diameter continuously increases in thethickness direction toward the surface of the membrane from the compactportion”.

A structure of the porous membrane in which a pore diameter continuouslyincreases in the thickness direction can be realized by, for example, amanufacturing method to be described later.

A maximum pore diameter of the porous membrane is preferably more than1.5 μm and 25 μm or less, is more preferably 1.8 μm to 23 μm, and iseven more preferably 2.0 μm to 21 μm. In the present specification, anaverage pore diameter of a section having the maximum average porediameter among section of the membrane cross section is referred to asthe “maximum pore diameter of the porous membrane”.

A ratio of the maximum pore diameter of the porous membrane to anaverage pore diameter of the compact portion (a ratio of a maximum porediameter to a minimum pore diameter of the porous membrane, which is avalue obtained by dividing the maximum pore diameter by the minimum porediameter, an “anisotropy ratio” in the present specification) ispreferably 3 or more, is more preferably 4 or more, and is even morepreferably 5 or more. The reason is that an average pore diameter exceptfor that of the compact portion increases to increase substancepermeability of the porous membrane. In addition, the anisotropy ratiois preferably 25 or less and is more preferably 20 or less. A ratio ofthe maximum pore diameter to the minimum pore diameter of the porousmembrane can be set to 3.0 to 20.0, for example.

It is preferable that a section line with a maximum average porediameter be a section closest to any surface of the membrane or asection in contact with that section.

In the section closest to any surface of the membrane, it is preferablethat an average pore diameter be more than 0.05 μm and 25 μm or less, bemore preferably more than 0.08 μm and 23 μm or less, and be even morepreferably more than 0.5 μm and 21 μm or less. In addition, a ratio ofan average pore diameter of the compact portion to an average porediameter of the section closest to any surface of the membrane ispreferably 1.2 to 20, is more preferably 1.5 to 15, and is even morepreferably 2 to 13.

(Elemental Distribution of Porous Membrane)

Formulas (I) and (II) are preferably satisfied for at least one surfaceof the porous membrane.B/A≤0.7  (I)A≥0.015  (II)

In the formula, A represents a ratio of an N element (nitrogen atom) toa C element (carbon atom) on a surface of the membrane, and B representsa ratio of the N element to the C element at a depth of 30 nm from thesame surface.

Formula (II) shows that a certain amount or more of N element is presenton at least one surface of the porous membrane, and Formula (I) showsthat an N element in the porous membrane is localized at a depth of lessthan 30 nm of the surface. The N element is preferably derived from anitrogen-containing polymer. In addition, the nitrogen-containingpolymer is preferably polyvinyl pyrrolidone.

With the surface satisfying Formulas (I) and (II), a bioaffinity of theporous membrane, particularly, a bioaffinity of the surface sidesatisfying Formulas (I) and (II) becomes high.

In the porous membrane, either one of surfaces may satisfy Formulas (I)and (II), or both surfaces may satisfy Formulas (I) and (II), but it ispreferable that both surfaces satisfy Formulas (I) and (II). In a casewhere either one of surfaces satisfies Formulas (I) and (II), thesurface thereof may be in an inside or an outside of a chamber fortransplantation to be described later, but the surface is preferably inthe inside thereof. In addition, in a case where only one of any surfacesatisfies Formulas (I) and (II), a surface satisfying Formulas (I) and(II) is preferably the surface X.

In the present specification, a ratio (A value) of N element to Celement on the membrane surface and a ratio (B value) of N element to Celement at a depth of 30 nm from the surface are obtained by calculatingusing XPS measurement results. The XPS measurement is X-rayphotoelectron spectroscopy, which is a method for irradiating a membranesurface with X-rays, measuring kinetic energy of photoelectrons emittedfrom the membrane surface, and analyzing a composition of elementsconstituting the membrane surface. Under conditions using amonochromated Al-Kα ray described in Examples, the A value is calculatedfrom results at the start of sputtering, and the B value is calculatedfrom time results, which are calculated that the ray is at 30 nm fromthe surface of the membrane measured from a sputtering rate.

It is sufficient for B/A to be 0.02 or more, and it is preferably 0.03or more, and is more preferably 0.05 or more.

A is preferably 0.050 or more and is more preferably 0.080 or more. Inaddition, it is sufficient for A to be 0.20 or less, and it ispreferably 0.15 or less, and is more preferably 0.10 or less.

It is sufficient for B to be 0.001 to 0.10, and it is preferably 0.002to 0.08, and is more preferably 0.003 to 0.07.

In a method for manufacturing the porous membrane which will bedescribed later, the elemental distribution of the porous membrane,especially the distribution of an N element, can be controlled by amoisture concentration contained in the temperature-controlled humidair, a time to apply the temperature-controlled humid air, a temperatureof a coagulation liquid, an immersion time, a temperature of adiethylene glycol bath for washing, an immersion time in the diethyleneglycol bath for washing, a speed of a porous manufacture line, and thelike. The distribution of the N element can also be controlled by anamount of moisture contained in a stock solution for forming a membrane.

(Composition of Porous Membrane)

The porous membrane contains a polymer. It is preferable that the porousmembrane be essentially composed of a polymer.

The polymer forming the porous membrane is preferably biocompatible. Theterm “biocompatible” means that the polymer has non-toxic andnon-allergenic properties, but does not have properties such that thepolymer is encapsulated in a living body.

The number average molecular weight (Mn) of the polymer is preferably1,000 to 10,000,000 and is more preferably 5,000 to 1,000,000.

Examples of polymers include thermoplastic or thermosetting polymers.Specific examples of polymers include polysulfone, cellulose acylate,nitrocellulose, sulfonated polysulfone, polyethersulfone,polyacrylonitrile, styrene-acrylonitrile copolymer, styrene-butadienecopolymer, saponified ethylene-vinyl acetate copolymer, polyvinylalcohol, polycarbonate, an organosiloxane-polycarbonate copolymer, apolyester carbonate, an organopolysiloxane, a polyphenylene oxide, apolyamide, a polyimide, polyamideimide, polybenzimidazole, ethylenevinyl alcohol copolymer, polytetrafluoroethylene (PTFE), and the like.From the viewpoints of solubility, optical physical properties,electrical physical properties, strength, elasticity, and the like,polymers may be homopolymers, copolymers, polymer blends, or polymeralloys.

Among these, polysulfone and cellulose acylate are preferable, andpolysulfone is more preferable.

The porous membrane may contain other components other than polymers asadditives.

Examples of additives include common salts, metal salts of inorganicacids such as lithium chloride, sodium nitrate, potassium nitrate,sodium sulfate, and zinc chloride; metal salts of organic acids such assodium acetate and sodium formate; polyethylene glycol; polymers such aspolyvinylpyrrolidone; polymer electrolytes such as sodium polystyrenesulfonate and polyvinyl benzyl trimethyl ammonium chloride; ionicsurfactants such as sodium dioctyl sulfosuccinate and sodium alkylsodium taurate; and the like. The additive may act as a swelling agentfor a porous structure.

The porous membrane is preferably a membrane formed from a singlecomposition as a single layer, and preferably not has a laminatedstructure of a plurality of layers.

(Method for Manufacturing Porous Membrane)

A method for manufacturing the porous membrane is not particularlylimited as long as the method can form the porous membrane having theabove-mentioned structure, and any general methods for forming a polymermembrane can be used. Examples of methods for forming a polymer membraneinclude a stretching method, a casting method, and the like.

For example, in the casting method, it is possible to produce a porousmembrane having the above-mentioned structure by adjusting the type andamount of a solvent used in a stock solution for forming a membrane, anda drying method after casting.

Manufacture of a porous membrane by a casting method can be carried outby a method including, for example, the following (1) to (4) in thisorder.

(1) A stock solution for forming a membrane, which contains a polymer,if necessary an additive and, if necessary a solvent, is flow-cast on asupport while being in a dissolved state.

(2) The surface of the flow-cast liquid membrane is exposed totemperature-controlled humid air.

(3) The membrane obtained after being exposed to temperature-controlledhumid air is immersed in a coagulation liquid.

(4) A support is peeled off if necessary.

A temperature of temperature-controlled humid air may be 4° C. to 60° C.and is preferably 10° C. to 40° C. A relative humidity of thetemperature-controlled humid air may be 30% to 70% and is preferably 40%to 50%. An absolute humidity of the temperature-controlled humid air ispreferably 1.2 to 605 g/kg air, and is more preferably 2.4 to 300 g/kgair. The temperature-controlled humid air may be applied at a wind speedof 0.1 μm/s to 10 μm/s for 0.1 seconds to 30 seconds, preferably 1second to 10 seconds.

In addition, an average pore diameter and position of the compactportion can also be controlled by a moisture concentration contained inthe temperature-controlled humid air and a time of applying thetemperature-controlled humid air. An average pore diameter of thecompact portion can also be controlled by an amount of moisturecontained in a stock solution for forming a membrane.

By applying the temperature-controlled humid air to the surface of theliquid membrane as described above, it is possible to cause coacervationfrom the surface of the liquid membrane toward the inside of the liquidmembrane by controlling evaporation of a solvent. By immersing themembrane in a coagulation liquid containing a solvent having lowsolubility of the polymer but compatible with the solvent of the polymerin this state, the above-mentioned coacervation phase is fixed as finepores, and pores other than the fine pores can also be formed.

A temperature of the coagulation liquid may be −10° C. to 80° C. in aprocess of immersing the membrane in the coagulation liquid. By changinga temperature during this period, it is possible to control a size of apore diameter up to a support surface side by adjusting a time from theformation of the coacervation phase on the support surface side to thesolidification from the compact portion. In a case where a temperatureof the coagulation liquid is raised, the formation of the coacervationphase becomes faster and a time for solidification becomes longer, andtherefore the pore diameter toward the support surface side tends tobecome large. On the other hand, in a case where a temperature of thecoagulation liquid is lowered, the formation of the coacervation phasebecomes slower and a time for solidification becomes shorter, andtherefore the pore diameter toward the support surface side is unlikelyto become large.

As the support, a plastic film or a glass plate may be used. Examples ofmaterials of the plastic film include polyester such as polyethyleneterephthalate (PET), polycarbonate, acrylic resin, epoxy resin,polyurethane, polyamide, polyolefin, a cellulose derivative, silicone,and the like. As the support, a glass plate or PET is preferable, andPET is more preferable.

The stock solution for forming a membrane may contain a solvent. Asolvent having high solubility of the polymer to be used (hereinafterreferred to as “favorable solvent”) may be used depending on a polymerto be used. As a favorable solvent, it is preferable that the solvent bequickly substituted with the coagulation liquid in a case where themembrane is immersed in the coagulation liquid. Examples of solventsinclude N-methyl-2-pyrrolidone, dioxane, tetrahydrofuran,dimethylformamide, dimethylacetamide, or a mixed solvent thereof in acase where the polymer is polysulfone and the like; dioxane,N-methyl-2-pyrrolidone, dimethylformamide, dimethylacetamide,dimethylsulfoxide, or a mixed solvent thereof in a case where thepolymer is polyacrylonitrile and the like; dimethylformamide,dimethylacetamide, or a mixed solvent thereof in a case where thepolymer is polyamide and the like; acetone, dioxane, tetrahydrofuran,N-methyl-2-pyrrolidone, or a mixed solvent thereof in a case where thepolymer is cellulose acetate and the like.

In addition to a favorable solvent, the stock solution for forming amembrane preferably use a solvent (hereinafter referred to as“non-solvent”) in which the solubility of the polymer is low but iscompatible with the solvent of the polymer. Examples of non-solventsinclude water, cellosolves, methanol, ethanol, propanol, acetone,tetrahydrofuran, polyethylene glycol, glycerin, and the like. Amongthese, it is preferable to use water.

A concentration of the polymer as the stock solution for forming amembrane may be 5% by mass to 35% by mass, is preferably 10% by mass to30% by mass. By setting the concentration thereof to 35 mass % or less,sufficient permeability (for example, water permeability) can beimparted to the obtained porous membrane. By setting the concentrationthereof to 5 mass % or more, the formation of a porous membrane whichselectively allows substances to permeate can be secured. An amount ofadditive to be added is not particularly limited as long as thehomogeneity of the stock solution for forming a membrane is not lost bythe addition, but is 0.5 mass % to 10 mass % respect to a generalsolvent. In a case where the stock solution for forming a membranecontains a non-solvent and a favorable solvent, a ratio of thenon-solvent to the favorable solvent is not particularly limited as longas a mixed solution can be maintained in a homogeneous state, but ispreferably 1.0% by mass to 50% by mass, is more preferably 2.0% by massto 30% by mass, and is even more preferably 3.0% by mass to 10% by mass.

As the coagulation liquid, it is preferable to use a solvent having alow solubility of the polymer used. Examples of such solvents includewater, alcohols such as methanol, ethanol, and butanol; glycols such asethylene glycol and diethylene glycol; aliphatic hydrocarbons such asether, n-hexane, and n-heptane; glycerol such as glycerin; and the like.Examples of preferred coagulation liquids include water, alcohols, or amixture of two or more of these. Among these, it is preferable to usewater.

After immersion in the coagulation liquid, it is also preferable toperform washing with a solvent different from the coagulation liquidthat has been used. Washing can be carried out by immersing in asolvent. Diethylene glycol is preferable as a washing solvent.Distribution of an N element in the porous membrane can be adjusted byadjusting either or both of a temperature and an immersion time ofdiethylene glycol in which a film is immersed by using diethylene glycolas a washing solvent. In particular, in a case wherepolyvinylpyrrolidone is used as the stock solution for forming amembrane of the porous membrane, a residual amount ofpolyvinylpyrrolidone on the membrane can be controlled. After washingwith diethylene glycol, furthermore, the membrane may be washed withwater.

As the stock solution for forming a membrane for the porous membrane, astock solution for forming a membrane which is obtained by dissolvingpolysulfone and polyvinylpyrrolidone in N-methyl-2-pyrrolidone, andadding water is preferable.

Regarding a method for manufacturing the porous membrane, reference canbe made to JP1992-349927A (JP-H04-349927A), JP1992-068966B(JP-H04-068966B), JP1992-351645A (JP-H04-351645A), JP2010-235808A, andthe like.

[Other Layers]

The immunoisolation membrane may contain layers other than the porousmembrane. Examples of other layers include a hydrogel membrane. As ahydrogel membrane, a biocompatible hydrogel membrane is preferable.Examples thereof include an alginic acid gel membrane, an agarose gelmembrane, a polyisopropyl acrylamide membrane, a membrane containingcellulose, a membrane containing a cellulose derivative (for example,methyl cellulose), a polyvinyl alcohol membrane, or the like. Thehydrogel membrane is preferably an alginic acid gel membrane. Specificexamples of alginic acid gel membranes include a polyion complexmembrane of alginic acid-poly-L-lysine-alginic acid.

<Method for Manufacturing Angiogenic Agent>

The angiogenic agent of the embodiment of the present invention can bemanufactured by a method including a step of enclosing mesenchymal stemcells with the immunoisolation membrane.

In the present invention, the immunoisolation membrane is used as aconstituent member of a chamber for transplantation which is forenclosing mesenchymal stem cells. The chamber for transplantation isused as a container for enclosing mesenchymal stem cells in a case oftransplanting the mesenchymal stem cells to a recipient. Theimmunoisolation membrane can be disposed on at least a part of thesurface forming an inner side and an outer side of the chamber fortransplantation. By disposing in such a manner, it is possible toprotect mesenchymal stem cells enclosed in the chamber fortransplantation from immune cells and the like present outside and tointroduce nutrients such as water, oxygen, and glucose into the insideof the chamber for transplantation from the outside.

The immunoisolation membrane may be disposed on the entire surface ofthe surface forming the inner side and the outer side of the chamber fortransplantation, and may be disposed a part of the surface correspondingto an area of, for example, 1% to 99%, 5% to 90%, 10% to 80%, 20% to 70%%, 30% to 60%, 40% to 50%, or the like with respect to the entire area.A surface on which the immunoisolation membrane is disposed may be onecontinuous portion or may be divided into two or more portions.

A shape of the chamber for transplantation is not limited, and may be ashape such as a pouched-like shape, a bag shape, a tube shape, amicrocapsule shape, or a drum shape. For example, a drum-shaped chamberfor transplantation can be formed by adhering the immunoisolationmembrane to the top and bottom of a silicone ring. A shape of thechamber for transplantation is preferably a shape capable of preventingmovement within a recipient in a case where the chamber fortransplantation is used as an angiogenic agent. Specific examples ofshapes of the chamber for transplantation include a cylindrical shape, adisk-like shape, a rectangular shape, an egg shape, a star shape, acircular shape, and the like. The chamber for transplantation may be ina form of a sheet, a strand, a spiral, or the like. The chamber fortransplantation may be a chamber for transplantation which encloses thecells or cell structure and becomes the above-described shape only in acase where the chamber for transplantation used as an angiogenic agent.

The chamber for transplantation may contain a biocompatible plastic orthe like for maintaining the shape and strength as a container. Forexample, the surface forming the inner side and the outer side of thechamber for transplantation may be made of an immunoisolation membraneand a biocompatible plastic that does not correspond to theimmunoisolation membrane. In addition, in the chamber fortransplantation in which the immunoisolation membrane is disposed on theentire surface of the surface forming the inner side and the outer side,a biocompatible plastic having a net-like structure may be furtherdisposed on the outside of the surface forming the inner side and theouter side, from the viewpoint of strength.

It is preferable in the chamber for transplantation that the surface Xof the porous membrane be on the inside thereof. That is, it ispreferable that the immunoisolation membrane be disposed so that thecompact portion of the porous membrane in the immunoisolation membraneis closer to the inside of the chamber for transplantation. By settingthe surface X in the inside of the chamber for transplantation, it ispossible to make permeability of physiologically active substanceshigher.

<Angiogenic Agent>

The angiogenic agent of the embodiment of the present invention includesmesenchymal stem cells and the immunoisolation membrane. In theangiogenic agent, mesenchymal stem cells are enclosed in theimmunoisolation membrane.

In the angiogenic agent, the immunoisolation membrane may enclose onlymesenchymal stem cells (or a cell structure of mesenchymal stem cells),or may enclose, in addition to the mesenchymal stem cells (or the cellstructure of mesenchymal stem cells), constituents or components otherthan the cell structure. For example, mesenchymal stem cells (or a cellstructure of mesenchymal stem cells) may be enclosed in theimmunoisolation membrane together with a hydrogel, and preferably in astate of being enclosed in the hydrogel. The angiogenic agent maycontain pH buffers, inorganic salts, organic solvents, proteins such asalbumin, or peptides.

The angiogenic agent may contain only one kind of mesenchymal stem cell(or a cell structure of mesenchymal stem cells) or may contain two ormore kinds thereof.

The angiogenic agent may be, for example, a device to beintraperitoneally or subcutaneously transplanted to a recipient. In thepresent specification, a recipient means a living body to whichtransplantation is performed. A recipient is preferably a mammal and ismore preferably a human.

With respect to the number of transplantation of the angiogenic agent ofthe embodiment of the present invention, the transplantation may beperformed once or the transplantation may be performed twice or more, asnecessary.

<Various Use Applications>

According to the present invention, there is provided an angiogenesismethod including a step of transplanting, to a subject in need ofangiogenesis, a cell transplant device including a mesenchymal stem cell(A) and an immunoisolation membrane (B) that encloses the mesenchymalstem cell.

According to the present invention, there is provided a cell transplantdevice which is used for angiogenesis procedure, the device including amesenchymal stem cell (A); and an immunoisolation membrane (B) thatencloses the mesenchymal stem cell.

According to the present invention, there is provided use of a celltransplant device to manufacture an angiogenic agent, in which the celltransplant device includes a mesenchymal stem cell (A); and animmunoisolation membrane (B) that encloses the mesenchymal stem cell.

In the various use applications described above, a preferable aspect isthe same as described above.

The present invention will be more specifically described using thefollowing examples, but is not limited by the examples.

EXAMPLES [Reference Example 1] Recombinant Peptide (Recombinant Gelatin)

The following CBE3 (which is disclosed in WO2008/103041A) was preparedas a recombinant peptide (recombinant gelatin).

CBE3:

Molecular weight: 51.6 kDa

Structure: GAP[(GXY)₆₃]₃G (SEQ ID NO: 11)

Number of amino acids: 571

Number of RGD sequences: 12

Imino acid content: 33%

Almost 100% of amino acids have a repeating structure of GXY In theamino acid sequence of CBE3, serine, threonine, asparagine, tyrosine,and cysteine are not included. CBE3 has an ERGD sequence (SEQ ID NO:10).

Isoelectric point: 9.34

GRAVY value: −0.682

1/IOB value: 0.323

Amino acid sequence (SEQ ID NO: 1 in a sequence listing) (which is thesame as that of SEQ ID NO: 3 in WO2008/103041A. However, X at the end iscorrected to “P”)

GAP(GAPGLQGAPGLQGMPGERGAAGLPGPKGERGDAGPKGADGAPGAPGLQGMPGERGAAGLPGPKGERGDAGPKGADGAPGKDGVRGLAGPIGPPGERGAAGLPGPKGERGDAGPKGADGAPGKDGVRGLAGPIGPPGPAGAPGAPGLQGMPGERGAAGLPGPKGERGDAGPKGADGAPGKDGVRGLAGPP)₃G

[Reference Example 2] Production of Porous Body of Recombinant Peptide

[PTFE-Thickness.Cylindrical Container]

A cylindrical cup-shaped polytetrafluoroethylene (PTFE) container with abottom-surface thickness of 3 mm, a diameter of 51 mm, a side-surfacethickness of 8 mm, and a height of 25 mm was prepared. In a case wherethe cylindrical cup has a curved surface as a side surface, the sidesurface is closed by PTFE with 8 mm and a bottom surface (circular shapeof a flat plate) is also closed by PTFE with 3 mm. In contrast, an uppersurface is in an open shape. Accordingly, an inner diameter of thecylindrical cup is 43 mm. Hereinafter, this container is referred to asa PTFE-thickness.cylindrical container.

[Aluminum Glass Plate-Cylindrical Container]

A cylindrical cup-shaped aluminum container with a thickness of 1 mm anda diameter of 47 mm was prepared. In a case where the cylindrical cuphas a curved surface as a side surface, the side surface is closed byaluminum with 1 mm and a bottom surface (circular shape of a flat plate)is also closed by aluminum with 1 mm. In contrast, an upper surface isin an open shape. In addition, TEFLON (registered trademark) with athickness of 1 mm is evenly spread only in the inside of the sidesurface, and as a result, an inner diameter of the cylindrical cup is 45mm. In addition, in the bottom surface of this container, a glass platewith 2.2 mm is bonded on the outside of aluminum. Hereinafter, thiscontainer is referred to as an aluminum glass plate cylindricalcontainer.

[Freezing Step in which Difference in Temperature is Small, and DryingStep]

A CBE3 aqueous solution was poured into the PTFE-thickness.cylindricalcontainer or the aluminum glass plate-cylindrical container, and wascooled down from the bottom surface using a cooling shelf within avacuum freeze dryer (TF5-85ATNNN: Takara Co., Ltd.). Combinations ofsetting of the container, a final concentration of the CBE3 aqueoussolution, an amount of the solution, and a temperature of the shelf atthis time were prepared as described below.

Condition A:

PTFE-thickness.cylindrical container, final concentration of CBE3aqueous solution of 4 mass %, and amount of aqueous solution of 4 mL. Asthe setting for the temperature of the shelf, the temperature was cooleddown until the temperature reached −10° C., and freezing was performedfor 1 hour at −10° C., for 2 hours at −20° C., for 3 hours at −40° C.,and finally for 1 hour at −50° C. Then, the frozen product was subjectedto vacuum drying for 24 hours at −20° C. after the setting of thetemperature of the shelf was returned to −20° C. After 24 hours, thetemperature of the shelf was increased to 20° C. in a state where thevacuum drying was continued as it was, and the vacuum drying was furtherperformed for 48 hours at 20° C. until a vacuum degree was sufficientlydecreased (1.9×10⁵ Pa). Then, the product was taken out of the vacuumfreeze dryer. Therefore, a porous body was obtained.

Condition B:

Aluminum-glass plate-cylindrical container, final concentration of CBE3aqueous solution of 4 mass %, and amount of aqueous solution of 4 mL.

As the setting for the temperature of the shelf, the temperature wascooled down until the temperature reached −10° C., and freezing wasperformed for 1 hour at −10° C., for 2 hours at −20° C., for 3 hours at−40° C., and finally for 1 hour at −50° C. Then, the frozen product wassubjected to vacuum drying for 24 hours at −20° C. after the setting ofthe temperature of the shelf was returned to −20° C. After 24 hours, thetemperature of the shelf was increased to 20° C. in a state where thevacuum drying was continued as it was, and the vacuum drying was furtherperformed for 48 hours at 20° C. until a vacuum degree was sufficientlydecreased (1.9×10⁵ Pa). Then, the product was taken out of the vacuumfreeze dryer. Therefore, a porous body was obtained.

Condition C:

PTFE-thickness.cylindrical container, final concentration of CBE3aqueous solution of 4 mass %, and amount of aqueous solution of 10 mL.As the setting for the temperature of the shelf, the temperature wascooled down until the temperature reached −10° C., and freezing wasperformed for 1 hour at −10° C., for 2 hours at −20° C., for 3 hours at−40° C., and finally for 1 hour at −50° C. Then, the frozen product wassubjected to vacuum drying for 24 hours at −20° C. after the setting ofthe temperature of the shelf was returned to −20° C. After 24 hours, thetemperature of the shelf was increased to 20° C. in a state where thevacuum drying was continued as it was, and the vacuum drying was furtherperformed for 48 hours at 20° C. until a vacuum degree was sufficientlydecreased (1.9×10⁵ Pa). Then, the product was taken out of the vacuumfreeze dryer. Therefore, a porous body was obtained.

[Measurement of Temperature in Each Freezing Step]

Regarding each of Conditions A to C, a liquid temperature of a surfaceof water in a circular center portion within a container was measured asa liquid temperature (non-cooled surface liquid temperature) of thefarthest portion from a cooling side in a solution, and a liquidtemperature of a bottom portion within the container was measured as aliquid temperature (cooled surface liquid temperature) of the closestportion to the cooling side in the solution.

As a result, each temperature and a profile of the difference intemperature are as shown in FIGS. 1 to 3.

It can be seen from FIGS. 1 to 3 that in Conditions A to C, the liquidtemperature fell below 0° C., which was a melting point, in a settingsection of a temperature of a shelf of −10° C. (before the temperaturewas decreased to −20° C.), and the solution was in a (unfrozen andovercooled) state where freezing did not occur in that state. Inaddition, in this state, the difference in temperature between thecooled surface liquid temperature and the non-cooled surface liquidtemperature was lower than or equal to 2.5° C. In the presentspecification, the “difference in temperature” means “non-cooled surfaceliquid temperature”−“cooled surface liquid temperature”. Then, thetiming at which the liquid temperature rapidly rose to around 0° C. byfurther decreasing the temperature of the shelf to −20° C. wasconfirmed, and it can be seen that freezing started due to generation ofsolidification heat at the timing. In addition, it was also possible toconfirm that ice formation actually started at this timing. Thereafter,the temperature was around 0° C. while the certain time passed. At thistime, the product was in a state where a mixture of water and ice waspresent. The temperature finally started to be decreased again from 0°C., and at this time, liquid disappeared and was changed to ice.Accordingly, the measured temperature was the solid temperature withinthe ice, that is, was not the liquid temperature.

Hereinafter, regarding Conditions A to C, the difference in temperaturein a case where the non-cooled surface liquid temperature reached amelting point (0° C.), the difference in temperature immediately beforethe temperature of the shelf was decreased from −10° C. to −20° C., andthe difference in temperature immediately before the generation ofsolidification heat are described. The “difference in temperatureimmediately before” referred to in the present invention indicates thehighest temperature within the difference in temperature which can bedetected between 1 second to 20 seconds before an event (such as thegeneration of solidification heat).

Condition A

Difference in temperature in a case where a non-cooled surface liquidtemperature reached a melting point (0° C.): 1.1° C.

Difference in temperature immediately before a temperature was decreasedfrom −10° C. to −20° C.: 0.2° C.

Difference in temperature immediately before generation ofsolidification heat: 1.1° C.

Condition B

Difference in temperature in a case where a non-cooled surface liquidtemperature reached a melting point (0° C.): 1.0° C.

Difference in temperature immediately before a temperature was decreasedfrom −10° C. to −20° C.: 0.1° C.

Difference in temperature immediately before generation ofsolidification heat: 0.9° C.

Condition C

Difference in temperature in a case where a non-cooled surface liquidtemperature reached a melting point (0° C.): 1.8° C.

Difference in temperature immediately before a temperature was decreasedfrom −10° C. to −20° C.: 1.1° C.

Difference in temperature immediately before generation ofsolidification heat: 2.1° C.

[Reference Example 3] Production of Biocompatible Polymer Block(Pulverization and Cross-Linking of Porous Body)

The CBE3 porous bodies of Conditions A and B which were obtained inReference Example 2 were pulverized by NEW POWER MILL (Osaka ChemicalCo., Ltd., NEW POWER MILL PM-2005). The pulverization was performed byone minute×5 times, that is, for 5 minutes in total at the maximumrotation speed. The obtained pulverized substances were sized with astainless steel sieve to obtain uncross-linked blocks with 25 to 53 μm,53 to 106 μm, and 106 to 180 μm. Then, the uncross-linked blocks weresubjected to thermal cross-linking (cross-linking was performed fortimes of six kinds of 8 hours, 16 hours, 24 hours, 48 hours, 72 hours,and 96 hours) at 160° C. under reduced pressure to obtainedbiocompatible polymer blocks (CBE3 blocks).

Hereinafter, a porous body-derived block of Condition A which iscross-linked for 48 hours is referred to as E, and a porous body-derivedblock of Condition B which is cross-linked for 48 hours is referred toas F. E and F are blocks with a small difference in temperature whichare formed from porous bodies produced through a freezing step in whichthe difference in temperature is small. Moreover, since the differencein cross-linking time did not influence the performance in theevaluation of the present examples, hereafter, the blocks cross-linkedfor 48 hours were used as a representative. There was no difference inthe performance between E and F. In Reference Examples, Examples, andComparative examples below, biocompatible polymer blocks which satisfiedCondition A, had sizes of 53 to 106 μm, and were produced with thecross-linking time of 48 hours were used.

[Reference Example 4] Measurement of Tap Density of BiocompatiblePolymer Block

The tap density is a value indicating how many blocks can be denselypacked in a certain volume, and it can be said that as the value becomeslower, the blocks cannot be densely packed, that is, the structure ofthe block is complicated. The tap density was measured as follows.First, a funnel with a cap (having a cylindrical shape with a diameterof 6 mm and a length of 21.8 mm: a capacity of 0.616 cm³) attached atthe tip thereof was prepared, and a mass of only the cap was measured.Then, the cap was attached to the funnel, and blocks were poured fromthe funnel so as to be accumulated in the cap. After pouring asufficient amount of blocks, the cap portion was hit 200 times on a hardobject such as a desk, the funnel was removed, and the blocks wereleveled off with a spatula. A mass was measured in a state where the capwas filled up with the blocks. The tap density was determined bycalculating a mass of only the blocks from the difference between themass of the cap filled up with the blocks and the mass of only the cap,and dividing the mass of only the blocks by the volume of the cap. As aresult, the tap density of the biocompatible polymer block of ReferenceExample 3 was 98 mg/cm³.

[Reference Example 5] Measurement of Cross-Linking Degree ofBiocompatible Polymer Block

The cross-linking degree (the number of times of cross-linking permolecule) of the blocks cross-linked in Reference Example 3 wascalculated. The measurement was performed by a TNBS(2,4,6-trinitrobenzene sulfonic acid) method.

<Sample Preparation>

A sample (about 10 mg), 4 mass % NaHCO₃ aqueous solution (1 mL), and 1mass % TNBS aqueous solution (2 mL) were added to a glass vial, and themixture was shaken for 3 hours at 37° C. Thereafter, 37 mass %hydrochloric acid (10 mL) and pure water (5 mL) were added thereto, andthen the mixture was allowed to stand for 16 hours or longer at 37° C.to prepare a sample.

<Preparation of Blank>

A sample (about 10 mg), 4 mass % NaHCO₃ aqueous solution (1 mL), and 1mass % TNBS aqueous solution (2 mL) were added to a glass vial, 37 mass% hydrochloric acid (3 mL) was immediately added thereto, and themixture was shaken for 3 hours at 37° C. Thereafter, 37 mass %hydrochloric acid (7 mL) and pure water (5 mL) were added thereto, andthen the mixture was allowed to stand for 16 hours or longer at 37° C.to prepare a blank.

The absorbances (345 nm) of the sample and the blank which were diluted10 times with pure water were measured, and the cross-linking degree(the number of times of cross-linking per molecule) was calculated from(Expression 2) and (Expression 3).(As−Ab)/14,600×V/w  (Expression 2)

(Expression 2) represents the amount (molar equivalent) of lysine per 1g of a recombinant peptide.

(In the expression, As represents a sample absorbance, Ab represents ablank absorbance, V represents an amount (g) of reaction liquid, and wrepresents a mass (mg) of the recombinant peptide.)1−(sample (Expression 2)/uncross-linked recombinant peptide (Expression2))×34  (Expression 3)

(Expression 3) represents the number of times of cross-linking permolecule.

As a result, the cross-linking degree of the biocompatible polymerblocks of Reference Example 3 was 4.2.

[Reference Example 6] Measurement of Water Absorption Rate ofBiocompatible Polymer Block

The water absorption rate of biocompatible polymer block produced inReference Example 3 was calculated.

A 3 cm×3 cm bag made of nylon mesh was filled with about 15 mg of thebiocompatible polymer block at 25° C., was swollen in ion exchange waterfor 2 hours, and then was dried with air for 10 minutes. The mass of thebag was measured at each stage, and the water absorption rate wasdetermined according to (Expression 4).Water absorption rate=(w2−w1−w0)/w0  (Expression 4)

(In the expression, w0 represents a mass of a material before waterabsorption, w1 represents a mass of an empty bag after water absorption,and w2 represents a mass of the whole bag containing the material afterwater absorption.)

As a result, the water absorption rate of the block of Reference Example3 was 786%.

[Reference Example 7] Production of Cell Structure

Mouse adipose-derived mesenchymal stem cells (mADSCs) were suspended ina D-MEM medium (Dulbecco's modified Eagle medium) containing 10% FBS(fetal bovine serum), the biocompatible polymer blocks (53 to 106 μm)produced in Reference Example 3 was added thereto, and finally, themADSCs (1.2×10⁸ cells) and the biocompatible polymer blocks (0.25 mg)suspended in 4 mL of the medium were seeded in EZSPHERE (registeredtrademark) dish Type 903 which is a cell non-adhesive dish of 35 mm(manufactured by AGC TECHNO GLASS CO., Ltd., a spheroid well diameter is800 μm, a spheroid well depth is 300 μm, the number of spheroid wells isabout 1,200, a bottom surface is a culture surface having recessedportions, and a side outer wall portion standing on the periphery of theculture surface is provided). The dish was allowed to stand for 48 hoursat 37° C. in a CO₂ incubator to obtain about 1,200 uniform cellstructures.

[Reference Example 8] Production of Immunoisolation Membrane andEvaluation of Pore Diameter

<Production of Porous Membrane>

15 parts by mass of polysulfone (P3500 manufactured by Solvay), 15 partsby mass of polyvinylpyrrolidone (K-30 manufactured by Nippon ShokubaiCo., Ltd.), 1 part by mass of lithium chloride, and 2 parts by mass ofwater were dissolved in 67 parts by mass of N-methyl-2-pyrrolidone.Thereby, a stock solution for forming a membrane was obtained. Thisstock solution for forming a membrane was cast on the surface of a PET(polyethylene terephthalate) film with a wet film thickness such that adry thickness became 50 μm. The flow-cast membrane surface was exposedto air adjusted to 30° C. and relative humidity 80% RH, at 2 m/sec for 5seconds. Immediately thereafter, the exposed membrane surface wasimmersed in a 65° C. coagulation liquid tank filled with water. The PETfilm was peeled off, and thereby a porous membrane was obtained.Thereafter, the porous membrane was put into a diethylene glycol bath at80° C. for 120 seconds, and then was washed with pure water. Thereby, aporous membrane having a dry thickness of 50 μm was obtained. Thisporous membrane was used as an immunoisolation membrane.

<Evaluation of Bubble Point>

In a pore diameter distribution measurement test using a permporometer(CFE-1200AEX manufactured by SEIKA CORPORATION), a bubble point of amembrane sample completely wetted by GALWICK (manufactured byPorousMaterials, Inc.) was evaluated after increasing an air pressure at5 cm³/min.

The bubble point of the porous membrane of Reference Example 8 was 0.58kg/cm².

<Evaluation of Thickness and Pore Diameter>

A thickness of the porous membrane was measured using a SEM photographof the cross section of the membrane.

Comparison of pore diameters in a thickness direction of the porousmembrane was performed by comparing pore diameters in 19 parting linesin a case where an SEM photograph of the membrane cross section wasdivided into 20 in the thickness direction of the membrane. 50 or moreconsecutive pores that intersect or are in contact with the parting lineare selected, each of pore diameters is measured, and an average valueis calculated as an average pore diameter. As the pore diameter, not alength of a portion where the selected pore intersects the parting line,but a diameter was used, the diameter being obtained by tracing a porewith a digitizer from the SEM photograph of the cross section of themembrane to calculate an area thereof, and calculating the obtained areaas an area of a true circle. In this case, for a parting line in whichpores are large and therefore only up to 50 pores can be selected, anaverage pore diameter is assumed to an average pore diameter obtained bymeasuring 50 pores by broadening the field of view of an SEM photographfor obtaining the membrane cross section. Pore diameters in thethickness direction of the membrane were compared by comparing theobtained average pore diameter for each parting line. In this case, thesmallest average pore diameter was defined as an average pore diameterof the compact portion.

A thickness of the compact portion of the porous membrane was calculatedfrom the following formula.Film thickness×[(number of parting lines that allows pore diameter bewithin 1.3 times minimum pore diameter)/19]  (Formula)

FIG. 4 illustrates a SEM photograph of the cross section of themembrane, and FIG. 5 illustrates a pore diameter distribution in thethickness direction. The results of evaluation of the thickness and porediameter of the porous membrane of Reference Example 8 were as follows.

A thickness of the porous membrane: 55 μm

An average pore diameter of the compact portion (an average value of theminimum pore diameter of the porous membrane): 0.8 μm

A ratio of the maximum pore diameter to the minimum pore diameter of theporous membrane: 7.5

A thickness of the compact portion: 10.5 μm

As shown in FIG. 5, in the porous membrane of Reference Example 8, theparting line Nos. 5 to 8 are compact portions, and based on thedefinition of paragraph number 0097 in the present specification, in theparting line Nos. 1 to 5 and the parting line Nos. 8 to 19, it can bedetermined that “the pore diameter continuously increases in thethickness direction toward the surface of the membrane from the compactportion”.

[Reference Example 9] Production of Cell Transplant Device(Immunoisolation Membrane Only)

The polysulfone porous membrane produced in Reference Example 8 was cutinto 3 cm×5 cm. The cut polysulfone porous membrane was folded into twosuch that a surface to which air was applied during the manufacturebecame an inner side.

Thereafter, using a sealer for tea bag (T-230K) manufactured byFUJIIMPULSE CO., LTD., a total of three sides of two long sides and oneshort side of a 3 cm×2.5 cm rectangle were heated to 260° C. Thetemperature was measured by a thermocouple. Thereafter, the remainingone side was inserted in a state where a metal rod was inserted intoIntramedic polyethylene tube (PE200), and in this state, both wererespectively heated at the same temperature using the same sealer.Thereafter, a surrounding portion was cut with a knife so that a widthof an end sealing portion became 1 mm. Thereby, a cell transplant device(only an immunoisolation membrane) having a size of 1 cm×2 cm wasproduced. The production method is shown in FIG. 6.

[Reference Example 10] Production of Immunoisolation Membrane andEvaluation of Pore Diameter

15 parts by mass of polysulfone (P3500 manufactured by Solvay), 15 partsby mass of polyvinylpyrrolidone (K-30 manufactured by Nippon ShokubaiCo., Ltd.), 1 part by mass of lithium chloride, and 2 parts by mass ofwater were dissolved in 67 parts by mass of N-methyl-2-pyrrolidone.Thereby, a stock solution for forming a membrane was obtained. Thisstock solution for forming a membrane was cast on the surface of a PETfilm with a wet film thickness such that a dry thickness became 83 μm.The flow-cast membrane surface was exposed to air adjusted to 30° C. andrelative humidity 57% RH, at 2 μm/sec for 5 seconds. Immediatelythereafter, the exposed membrane surface was immersed in a 70° C.coagulation liquid tank filled with water. The PET film was peeled off,and thereby a porous membrane was obtained. Thereafter, the porousmembrane was put into a diethylene glycol bath at 80° C. for 120seconds, and then was washed with pure water. Thereby, a porous membranewas obtained. This porous membrane was used as an immunoisolationmembrane.

The bubble point of the porous membrane of Reference Example 10 was 0.66kg/cm².

A thickness and a pore diameter were evaluated as in the same manner asReference Example 8. FIG. 7 illustrates a SEM photograph of the crosssection of the membrane, and FIG. 8 illustrates a pore diameterdistribution in the thickness direction. The results of evaluation ofthe thickness and pore diameter of the porous membrane of ReferenceExample 10 were as follows.

A thickness of the porous membrane: 85 μm

An average pore diameter of the compact portion (an average value of theminimum pore diameter of the porous membrane): 0.73 μm

A ratio of the maximum pore diameter to the minimum pore diameter of theporous membrane: 11.1

A thickness of the compact portion: 21.8 μm

As shown in FIG. 8, in the porous membrane of Reference Example 10, theparting line Nos. 4 to 8 are compact portions, and based on thedefinition of paragraph number 0097 in the present specification, in theparting line Nos. 1 to 4 and the parting line Nos. 8 to 19, it can bedetermined that “the pore diameter continuously increases in thethickness direction toward the surface of the membrane from the compactportion”.

[Reference Example 11] Production of Cell Transplant Device

A cell transplant device was produced in the same manner as in ReferenceExample 9 using the polysulfone porous membrane produced in ReferenceExample 10 instead of the polysulfone porous membrane produced inReference Example 8 as the porous membrane.

[Reference Example 12] Production of Immunoisolation Membrane andEvaluation of Pore Diameter

18 parts by mass of polysulfone (P3500 manufactured by Solvay), 12 partsby mass of polyvinylpyrrolidone (K-30), 0.5 parts by mass of lithiumchloride, and 1 parts by mass of water were dissolved in 68.5 parts bymass of N-methyl-2-pyrrolidone. Thereby, a stock solution for forming amembrane was obtained. This stock solution for forming a membrane wascast on the surface of a PET film with a wet film thickness such that adry thickness became 130 μm. The flow-cast membrane surface was exposedto air adjusted to 30° C. and relative humidity 50% RH, at 2 μm/sec for5 seconds. Immediately thereafter, the exposed membrane surface wasimmersed in a 50° C. coagulation liquid tank filled with water. The PETfilm was peeled off, and thereby a porous membrane was obtained.Thereafter, the porous membrane was put into a diethylene glycol bath at80° C. for 120 seconds, and then was washed with pure water. Thereby, aporous membrane was obtained. This porous membrane was used as animmunoisolation membrane.

The bubble point of the porous membrane of Reference Example 12 was 1.3kg/cm².

A thickness and a pore diameter were evaluated as in the same manner asReference Example 8. FIG. 9 illustrates a SEM photograph of the crosssection of the membrane, and FIG. 10 illustrates a pore diameterdistribution in the thickness direction. The results of evaluation ofthe thickness and pore diameter of the porous membrane of ReferenceExample 2 were as follows.

A thickness of the porous membrane: 142 μm

An average pore diameter of the compact portion (an average value of theminimum pore diameter of the porous membrane): 0.45 μm

A ratio of the maximum pore diameter to the minimum pore diameter of theporous membrane: 12.2

A thickness of the compact portion: 27.4 μm

As shown in FIG. 10, in the porous membrane of Reference Example 12, theparting line Nos. 2 to 5 are compact portions, and based on thedefinition of paragraph number 0097 in the present specification, in theparting line Nos. 1 and 2 and the parting line Nos. 5 to 19, it can bedetermined that “the pore diameter continuously increases in thethickness direction toward the surface of the membrane from the compactportion”.

[Reference Example 13] Production of Cell Transplant Device(Immunoisolation Membrane Only)

A cell transplant device (immunoisolation membrane only) was produced inthe same manner as in Reference Example 9 using the polysulfone porousmembrane produced in Reference Example 12 instead of the polysulfoneporous membrane produced in Reference Example 8 as the porous membrane.

[Example 1] Evaluation of Ability to Induce Blood Vessels Around DeviceIn Vivo

1,600 cell structures of mADSC produced in Reference Example 7 weresealed in the cell transplant devices (only an immunoisolation membrane)produced in Reference Examples 9, 11, and 13, and an injection part wassealed to complete the cell transplant devices (angiogenic agents). Theywere implanted subcutaneously in the back of a NOD/SCID mouse, and after2 weeks, a tissue section of the transplantation site was produced toperform histological evaluation. A representative tissue specimentransplanted into two individuals is shown in FIG. 11. As a result, itwas found that many new blood vessels were locally induced in thevicinity of the cell transplant device including the cell structure of amesenchymal stem cell (MSC).

[Comparative Example 1] Evaluation of Ability to Induce Blood VesselsAround Device In Vivo

Only the cell structure of mADSC produced in Reference Example 7 wasimplanted subcutaneously on the back of a NOD/SCID mouse, and after 2weeks, a tissue section of the transplantation site was produced toperform histological evaluation. A representative tissue specimentransplanted into two individuals is shown in FIG. 12. As a result, newblood vessels were induced at random sites in the case of only the cellstructure of a mesenchymal stem cell (mADSC).

[Comparative Example 2] Evaluation of Ability to Induce Blood VesselsAround Device In Vivo

The cell transplant devices produced in Reference Examples 9, 11, and 13were implanted subcutaneously on the back of a NOD/SCID mouse, and after2 weeks, tissue sections of the transplantation sites were produced toperform histological evaluation. A representative tissue specimentransplanted into two individuals is shown in FIG. 13. As a result, itwas found that an amount of new blood vessels induced was very small inthe cell transplant device not including the cell structure of amesenchymal stem cell (mADSC).

Example 2

The results of Example 1 and Comparative Examples 1 and 2 werequantitatively evaluated based on a total area of blood vessels pervisual field and the number of blood vessels per visual field. In theevaluation, data of 4 individuals were analyzed, and an average valueand a standard deviation were calculated. As a result, in thetransplantation results of the “cell structure+cell transplant device”(Example 1), in the vicinity of the device, a total area of blood vesselper visual field was 13,391±4,329 μm², and the number of blood vesselsper visual field was 28.5±7.0. On the other hand, in the transplantationresults of the “cell structure only” (Comparative Example 1), a totalarea of blood vessel per visual field was 1,571±1,264 μm², and thenumber of blood vessels per visual field was 8.0±3.2. According to thetransplantation results of the “only the cell transplant device”(Comparative Example 2), a total area of blood vessel per visual fieldwas 3,519±3,826 μm², and the number of blood vessels per visual fieldwas 8.5±7.1. Based on these results, it became clear that, in thequantitative determination, the “cell structure+cell transplant device”induces a significantly large amount of new blood vessels around thedevice as compared to the “only the cell structure” or “only the celltransplant device” (refer to FIGS. 14 to 17). Regarding statisticalanalysis, by the t-test, it was found that there is a significantdifference between the evaluation results of the total area of bloodvessel and the evaluation results of the number of blood vessels betweenthe “only the cell structure” or “only the cell transplant device” andthe “cell structure+cell transplant device” (p<0.05).

[Example 3] Evaluation of Ability to Induce Blood Vessels Around DeviceIn Vivo (Different Recipient Animals)

1,600 cell structures of mADSC produced in Reference Example 7 weresealed in the cell transplant device (only an immunoisolation membrane)produced in Reference Example 9, and an injection part was sealed tocomplete the cell transplant device. They were implanted subcutaneouslyin the back of a C57BL/6 mouse, and after 2 weeks, a tissue section ofthe transplantation site was produced to perform histologicalevaluation. In addition, a tissue specimen compared with the case whereonly the cell transplant device produced in Reference Example 9 wastransplanted is shown in FIG. 18. As a result, it was found that, in thecell transplant device including the cell structure, even in a case oftransplantation to the C57BL/6 mouse, a larger number of new bloodvessels were induced in the vicinity of the device as compared to thecase where the cell transplant device (an immunoisolation membrane only)was transplanted.

[Reference Example 14] Preparation of Spheroid

1.2×10⁸ cells of mouse adipose-derived mesenchymal stem cells (mADSCs)suspended in 4 mL of a D-MEM medium (Dulbecco's modified Eagle medium)containing 10% FBS (fetal bovine serum) were seeded in EZSPHERE(registered trademark) dish Type 903 which is a cell non-adhesive dishof 35 mm (manufactured by AGC TECHNO GLASS CO., Ltd., a spheroid welldiameter is 800 μm, a spheroid well depth is 300 μm, the number ofspheroid wells is about 1,200, a bottom surface is a culture surfacehaving recessed portions, and a side outer wall portion standing on theperiphery of the culture surface is provided). The dish was allowed tostand for 48 hours at 37° C. in a CO₂ incubator to obtain about 1,200uniform spheroids.

[Example 4] Evaluation of Ability to Induce Blood Vessels Around DeviceIn Vivo

1,600 spheroids of mADSC produced in Reference Example 14 were sealed inthe cell transplant device (only an immunoisolation membrane) producedin Reference Example 9, and an injection part was sealed to complete thecell transplant device (an angiogenic agent). They were implantedsubcutaneously in the back of a C57BL/6 mouse, and after 2 weeks, atissue section of the transplantation site was produced to performhistological evaluation. A representative tissue specimen transplantedinto two individuals is shown in FIG. 19. As a result, it was found thatnew blood vessels were induced in the vicinity of the cell transplantdevice including mesenchymal stem cells (MSCs).

Example 5

The results of Example 4 were quantitatively evaluated based on a totalarea of blood vessels per visual field and the number of blood vesselsper visual field. In the evaluation, data of 4 individuals wereanalyzed, and an average value and a standard deviation were calculated.As a result, in the transplantation results of the “cells+celltransplant device” (Example 4), a total area of blood vessel per visualfield was 5871±1053 μm², and the number of blood vessels per visualfield was 16±5.6. Based on the results of Example 2 and Example 4, itbecame clear that the “cells+cell transplant device” induces a largeamount of new blood vessels as compared to the “only the cell transplantdevice” (refer to FIGS. 20 and 21).

SEQUENCE LISTING

International Application 17F02810W1 Angiogenic Agent and Method forManufacturing Same and JP1803216220180830-00210410351801780099 Normal20180830145537201808061055230470_P1AP101_17_0.app Based on InternationalPatent Cooperation Treaty

What is claimed is:
 1. An angiogenesis method which comprisestransplanting, to a subject in need of angiogenesis, a cell transplantdevice including a mesenchymal stem cell (A) and an immunoisolationmembrane (B) that encloses the mesenchymal stem cell, wherein theimmunoisolation membrane is a porous membrane comprising a thermoplasticor thermoset polymer and wherein within an inner side of the porousmembrane, a layered compact portion in which a pore diameter isminimized is present, and the pore diameter continuously increases as agradient in a thickness direction from the compact portion toward atleast one surface of the porous membrane, wherein the porous membrane isa membrane formed from a single composition as a single layer.
 2. Theangiogenesis method according to claim 1, wherein the mesenchymal stemcell is an adipose-derived mesenchymal stem cell or abone-marrow-derived mesenchymal stem cell.
 3. The angiogenesis methodaccording to claim 1, wherein the thermoplastic or thermoset polymer isselected from polysulfone or polyvinylpyrrolidone.
 4. The angiogenesismethod according to claim 3, wherein a minimum pore diameter of theporous membrane is 0.02 μm to 1.5 μm.
 5. The angiogenesis methodaccording to claim 3, wherein a thickness of the porous membrane is 10μm to 250 μm.
 6. The angiogenesis method according to claim 3, wherein aratio of a maximum pore diameter to a minimum pore diameter of theporous membrane ranges from 3:1 to 20:1.
 7. The angiogenesis methodaccording to claim 1, wherein a thickness of the compact portion is 0.5μm to 30 μm.
 8. The angiogenesis method according to claim 1, whereinthe mesenchymal stem cell is contained as a cell structure whichincludes a plurality of biocompatible polymer blocks and a plurality ofmesenchymal stem cells of at least one type, and in which at least oneof the biocompatible polymer blocks is disposed in gaps between theplurality of mesenchymal stem cells.
 9. The angiogenesis methodaccording to claim 8, wherein a size of one of the biocompatible polymerblocks is 20 μm to 200 μm.
 10. The angiogenesis method according toclaim 8, wherein, in the biocompatible polymer blocks, a biocompatiblepolymer is cross-linked by heat, ultraviolet rays, or an enzyme.
 11. Theangiogenesis method according to claim 8, wherein the biocompatiblepolymer blocks have an amorphous shape.
 12. The angiogenesis methodaccording to claim 8, wherein the cell structure includes 0.0000001 μgto 1 μg of the biocompatible polymer blocks per cell.